CONCACAF Champions League Wrap: Pachuca miss out on top seeding
The Mexicans needed a win to be ranked top, only to suffer a shock 3-2 loss at Real Espana in their final Group One game.
Enrique Meza's men still topped the group on nine points, while the Hondurans recorded their first win of the campaign.
Eder Delgado headed in a Camilo Aguirre cross in the third minute to put Real Espana ahead.
But their lead only lasted six minutes as Simon Steven Almeida's low strike from inside the area found the bottom corner.
It was 2-2 at half-time as Aviles Hurtado cancelled out Kevin Espinoza's goal before Delgado's second in the 64th minute proved to be decisive.
DC United are now guaranteed the top seeding.
The Montreal Impact had already sealed top spot in Group Three before a 1-1 draw against the New York Red Bulls.
Jack McInerney tapped in an Andres Romero cross to give the Impact the lead in the 71st minute, but Connor Lade equalised for New York.