The New York Jets needed a win against the Buffalo Bills to secure a play-off berth, but suffered a 22-17 defeat on Sunday.
The Jets had the chance to qualify for the post-season for the first time in five seasons, but former coach Rex Ryan came back to haunt his old side.
With the Pittsburgh Steelers beating the Cleveland Browns 28-12, they moved ahead of the Jets and secured the AFC's final wild-card spot.
Ben Roethlisberger threw three touchdown passes, with the Steelers pulling away from the Browns in the fourth quarter for a big win.
The Denver Broncos earned home-field advantage in the play-offs with a 27-20 win over the San Diego Chargers, clinching the top seed in the AFC.
A 20-point haul after half-time from the Broncos, aided by field position and a late interception by the defence, helped them to victory.
The New England Patriots fell to number two seed after they suffered a 20-10 loss to the Miami Dolphins.
The Cincinnati Bengals (24-16 win over the Baltimore Ravens) are next in the order, followed by the Houston Texans (30-6 win over the Jacksonville Jaguars), the Kansas City Chiefs (23-17 win over the Oakland Raiders) and Steelers.
In the NFC, the Carolina Panthers secured the top seed after 38-10 win over the Tampa Bay Buccaneers.
The Arizona Cardinals are the second seed despite a 36-6 loss to the Seattle Seahawks.
The Washington Redskins are locked in at number four after a 34-23 win over the Dallas Cowboys, and the third, fifth and sixth places were decided by the last game of the night between the Minnesota Vikings and the Green Bay Packers.
A 20-13 win for the Vikings ensured they finish as the third seed, with the Packers finishing as number five, with the Seahawks finishing as sixth seed.
In other results, the New Orleans Saints got up 20-17 against the Atlanta Falcons, the Indianapolis Colts sealed a 30-24 win against the Tennessee Titans, and the Detroit Lions defeated the Chicago Bears 24-20.
Meanwhile, the Philadelphia Eagles edged the New York Giants 35-30 and Jim Tomsula was sacked as coach of the San Francisco 49ers after one season following their 19-16 overtime win against the St. Louis Rams.