Chiefs kicker Ryan Succop missed a 41-yard field goal attempt with four seconds left in regulation, a kick that could have eliminated the Chargers while vaulting the Pittsburgh Steelers into the playoffs.
San Diego beat Kansas City 27-24 in overtime to clinch the AFC's second wild card.
But based on a new rule this season, the Chargers should have been flagged for illegal formation prior to Succop's missed field goal, a 5-yard penalty that would have given Kansas City a 36-yard game-winning attempt.
"On the play, San Diego lined up with seven men on one side of the snapper," the league said in a statement released Monday morning. "This should have been penalized as an illegal formation by the defense."
The rule (Rule 9, Section 1, Article 3) is in relation to the overloading of one side of the formation when defending a field goal. Replays clearly showed the Chargers had seven players lined up on the right side of the defensive formation.
"The rule was adopted this year as a player safety measure," the league's statement said. "The penalty for illegal formation by the defense is a loss of five yards.
"This rule is not subject to instant replay review. Had the penalty been assessed, it would have resulted in a fourth-and-7 from the San Diego 18 with 0:04 remaining, enabling the Chiefs to attempt a 36-yard field goal."
Had the Chargers lost Sunday, the Steelers (8-8) would have claimed the No. 6 seed in the AFC playoffs based on conference tiebreakers.
Instead, San Diego (9-7) will visit the Cincinnati Bengals on Sunday in the wild-card round.
ESPN.com Chargers reporter Eric D. Williams contributed to this report.