At the time of James V's death in 1542, Lennox possessed a strong claim to the throne of Scotland should Mary Queen of Scots, then an infant, pass away without an heir. He was James V's second cousin once removed, being a great-grandson of Princess Mary, James II's daughter, through her daughter, Elizabeth Hamilton. However, the Earl of Arran, James Hamilton, was descended from a son of Princess Mary, as thus held the stronger claim. As a result, Lennox was bumped to third in line of succession.
He spent most of his youth in exile in England, but returned to assert his claims to the line of succession when James V died. In 1544 he married Margaret Douglas (1515-1578), half-sister of the previous King James V of Scotland. This significantly improved the claim to the Scottish throne of any of his progeny, and also introduced a claim to the English throne. Alas, his return to Scotland was short-lived; he supported Henry VIII's military efforts to secure a marriage between Mary Queen of Scots and his son Edward, Prince of Wales in the Rough Wooing. He fled once more to England with his wife.
Returned to Scotland upon Elizabeth I's urging during Mary Queen of Scots's marriage negotiations in 1564. He quickly took up his position as the most powerful lord in the Glasgow area and played a vital role in the turbulent years to come.
Whether or not Elizabeth's intention was for Lennox's beautiful son, Lord Darnley, to capture Mary's heart (and eliminate the possibility of the Scottish Queen making a powerful marriage to a continental - or worse, Catholic - prince) is a matter of conjecture. In any event, Lennox was instrumental in the marriage of his son to Mary I of Scotland. Elizabeth reacted with disapproval of the marriage because Darnley, technically born in England and thus one of her subjects, had married without her permission. She then threw Lennox's wife into the Tower of London.
After Darnley's 1567 murder, Lennox became the most ardent pursuant of justice against the lords who had conspired in the murder. He also became the star witness against Mary, though her involvement in the murder, most certainly carried out by her then lover and later husband, Lord Bothwell, is controversial.