Born in 1551 as the son of Sogo Kazumasa, nephew of the great Miyoshi Nagayoshi, Yoshitsugu was raised by his uncle after his father's sudden death in 1561. When his cousin Miyoshi Yoshioki died young in 1563, he was adopted as Nagayoshi's heir and took the Miyoshi surname; upon Nagayoshi's death in 1564 he became head of the Miyoshi clan while still a boy. In May 1565 he received a character from shogun Ashikaga Yoshiteru's name and was renamed 'Yoshishige,' but only nineteen days later he joined the Miyoshi Triumvirate and Matsunaga Hisamichi in the Eiroku Incident, storming the Nijo Palace and killing Yoshiteru; he then took the name 'Yoshitsugu.' When the Triumvirate installed Ashikaga Yoshihide as a rival shogunal figure and began to marginalize him, Yoshitsugu allied with Matsunaga Hisahide in 1567 to counter them. In 1568 he supported Oda Nobunaga's march on Kyoto and was confirmed in his holdings of northern Kawachi Province and Wakae Castle. Around 1571 he turned against Nobunaga and joined the anti-Nobunaga coalition, and in July 1573 he sheltered the exiled shogun Ashikaga Yoshiaki at Wakae Castle. Enraged, Nobunaga sent a large force under Sakuma Nobumori against Wakae Castle; betrayed by his own retainers, the 'Three Men of Wakae,' the castle fell in November 1573. Yoshitsugu killed his wife and children, then took his own life by cutting his belly crosswise. With his death, the Miyoshi main line — which had once ruled eight provinces across the Kinai and Shikoku — came to an end.

Historians remain divided on his character. Imatani Akira harshly judged him as possessing "ability inferior in every way" to his uncle Nagayoshi, while Amano Tadayuki views him as a reformer who sought to "overcome" the authority of the Ashikaga shogunate, even if he lacked the practical power to realize his vision. His dramatic final stand drew admiration even from contemporaries: Ota Gyuichi, author of the Shincho Koki, called it "a peerless feat, and a piteous sight," and modern historian Sudo Shigeki has similarly praised it as "a magnificent end."

Yoshitsugu's death marked the definitive end of the Miyoshi regime that his uncle Miyoshi Nagayoshi had built across eight provinces of the Kinai and Shikoku. Wakae Castle in present-day Higashiosaka, where he made his last stand, was later entrusted to the 'Three Men of Wakae' and used as a siege fort during the Ishiyama War before being abandoned in 1580. Together with Furuhashi Castle in Kadoma, which his forces had earlier defended, it remains a historical site testifying to the rise and fall of the Miyoshi clan in the Kinai region.