Hamato Yoshi was an orphan, who, along with his best friend Yukio Mashimi, begged in the streets of 1960s Japan. When Yoshi returned a 5-yen coin dropped by the Ancient One, he took the two of them in for his honesty, and raised them both as his sons, training them in the art of Ninjutsu. The two boys grew up together in the same house, and learning the same arts. Unfortunately, they both also loved the same girl, Tang Shen, another orphan the Ancient One had taken in. Tang Shen loved Yoshi more. During this time, Tang Shen spared a rat that had sneaked into the house, and took it as a pet. One night, Yoshi, Shen, and Mashimi observed a lone man being attacked by four ninja. The two young men decided to help him, and fought off the ninja. The man they saved was none other than the Utrom Mortu, and the ninja were members of the Foot Clan. Impressed by their skill, and having been partially exposed when his robotic arm was sliced off in the battle, Mortu gave Yoshi and Mashimi the offer to become Guardians, which they both accepted over the objections of their master.
As a Guardian, Yoshi was promoted time and again, but Mashimi never seemed to advance. Mashimi grew jealous as Yoshi surpassed him in every way, and in his anger, he murdered Tang Shen and betrayed the Utroms to Ch'rell. Yoshi and the Utroms escaped. When Yoshi discovered what had happened he went into a rage, stormed the Foot's citadel and slew Mashimi in single combat. With their Tokyo headquarters compromised, the Utroms relocated to New York, under the name of TCRI. Yoshi went with them, taking along Tang Shen's pet rat, which he named Splinter, to remind himself of what he had done and what he had failed to do..
The Shredder eventually tracked Yoshi down, and tried to force him to reveal the location of the Utroms. Yoshi resisted, and was killed for his defiance. Splinter escaped and fled to the sewers, where he mutated along with the Turtles. He then proceeded to teach ninjutsu to the Turtles as he himself had learned it from Yoshi. In the "lost season" of the series, it is learned that Yoshi and the Ancient One had tried, in vain, to get the Ninja Tribunal's help in the fight against the Utrom Shredder. They refused as he was not the 'true' Shredder even though the Utrom Shredder was growing in power. Due to their refusal to help, Splinter partly blamed the Ninja Tribunal for Yoshi's death.
Yoshi, a former Battle Nexus champion, is seen in flashbacks of the history of the Battle Nexus. A statue of him is in the hall of champions of the Nexus, and next to him are statues of Splinter and Michelangelo, who won the following two tournaments. Before the Utroms departed Earth, they gave Splinter an orb that contained a hologram of Yoshi. That orb was destroyed by the Utrom Shredder's adoptive daughter, Karai, when she attacked the Turtles' lair in the episode "Scion of the Shredder".
In the "lost" season five's final episode "Enter the Dragons Part II", a spirit of Hamato Yoshi appears, summoned by the combined power of the Turtles' dragon avatars, and defeats the Tengu Shredder. After delivering the final blow against the Tengu Shredder, he exchanges a brief greeting with Splinter before he disappears.