The Doom Sub is a large, metallic, Plesiosaur-like submarine owned by King Bowser Koopa, that was only ever seen on The Adventures of Super Mario Bros. 3. Whereas the Doom Ship was built for travel above land, the Doom Sub was created specifically for aquatic attacks. Although its origin was never explained, it can be inferred that the Doom Sub was built by Kooky von Koopa, as he was the one who built the Doom Ship. Despite appearing prominently in two separate episodes, the Doom Sub is never referenced in any other form of Mario media.
The Doom Sub has a number of special features and abilities. Like the Doom Ship, the Doom Sub is equipped with special holes for Rocky Wrenches to hide in and attack from. It is also capable of launching Bob-ombs and Nasty Nets at unwanted targets. The Doom Sub can also blast mysterious lasers, which have been shown to magically encase their target in a block of cement.
The Doom Sub first appears in the episode "The Ugly Mermaid", where Bowser, accompanied by Hip and Hop Koopa, use it to try and take over the underwater city of Mertropolis. Mario, Luigi, Princess Toadstool, and Toad pursue the Doom Sub to Mertropolis, where they are bombarded by Rocky Wrenches. Mario and Luigi use the wrenches initially thrown by the Rocky Wrenches to retaliate against the Doom Sub's attacks, and knock it out of control by hitting the rotors. Bowser then activates the Doom Sub's lasers, one of which hits Mario, and encases him in a block of cement, nearly drowning him.
Bowser soon announces that he is going to destroy Mertropolis within twenty minutes with the Doom Sub if the citizens refuse to hand over their city to him. With Mario too nervous to go back in the water, Luigi, Princess Toadstool and Toad attempt to stop the Doom Sub on their own, only to get caught in Bowser's Nasty Nets. As the people of Mertropolis fail to surrender, Bowser drops a Bob-omb from the Doom Sub onto Mertropolis' dome, which blows up and starts to flood the city. Mario gets over his fear of drowning so that he can seal up the hole and rescue his friends. After the rescue, Mario and Luigi use one of the Nasty Nets to gather a school of Jelectros, which they use to electrocute the entire Doom Sub, blasting it away from Mertropolis.
The Doom Sub then reappears in the episode "The Venice Menace". Mario and his friends first hear about the Doom Sub when a gondolier from Venice appears in Desert Land, shouting about how a "Sea Monster" is attacking his home town. Although Mario and Luigi head for Venice to stop the Sea Monster, they have no knowledge that it is actually the Doom Sub. It turns out that Bowser and Kootie Pie Koopa are using the Doom Sub to scare everyone out of Venice so that they can warp the city back to Dark Land and turn it into the "Kootie Pie Koopa Water Park".
Not long after Mario and Luigi arrive in Venice, they are spotted and chased around by the Doom Sub. Using a motorboat, Mario and Luigi make it to dry land, where the Doom Sub crashes and gets a leak in one of its walls, which Kootie Pie has to fix with a small Fire Bro. To get back at the Mario Bros., Bowser deploys several Boss Basses and Chain Chomps to destroy the foundations of several buildings, sinking them. One of the buildings is the hotel that Mario and Luigi are sleeping in. However, Mario and Luigi evade the minions that attack them, change into their Frog Suits, and go after the Doom Sub. While Mario distracts Bowser and Kootie Pie by luring them into the Doom Sub's engine room, Luigi pulls the Doom Sub's recall lever, and recalls all the minions that had been unleashed on Venice. After returning to the helm, Mario pulls another lever, and leaves the Doom Sub with Luigi, just as the entire Sub warps itself to Desert Land. The impact of the warp damages the Doom Sub, which in turn ruins Bowser's plan, and sends Kootie Pie into tears and causes her to throw a temper tantrum.