The 2016 Lamar Hunt U.S. Open Cup tournament proper will feature teams from all five tiers of men's soccer of the American Soccer Pyramid.
Starting with the 2016 tournament, US Soccer took over the qualifying process that used to be handled by each association. According to US Soccer, all teams within the Division I, II, & III professional leagues will qualify automatically as in past years. Any national league not in the top three divisions can apply to use previous year's league standings as their qualification method. Remaining teams will participate in up to five qualifying rounds to determine entrants into the tournament proper. In prior years, slots were allocated and the individual associations created tournaments and qualifiers to determine their representatives. Final slot allocation will be determined when team registration has concluded.
As of September 8, 2015, US Soccer has made the decision to ban all lower division teams that are majority owned by a higher division team. This means Bethlehem Steel, LA Galaxy 2, New York Red Bulls 2, Orlando City B, Portland Timbers 2, Real Monarchs, Seattle Sounders 2, and Swope Park Rangers (all from the United Soccer League) will not qualify for the 2016 tournament. On November 5, 2015, New York Cosmos Chief Operating Officer Erik Stover stated that eligible New York Cosmos B would not enter as "the integrity of the tournament is more important". Just before the tournament, USL side Rio Grande Valley FC Toros and their MLS parent Houston Dynamo asked that RGVFC be banned from the tournament by US Soccer. While RGVFC is not owned by the Dynamo, the MLS side staffs and coaches the Toros.
On February 3, teams scheduled for the Third Qualifying Round received letters from USSF advising them that due to the withdrawal of some professional teams (the USL clubs mentioned above), the round was cancelled and all twelve teams had qualified for the First Round of the main tournament along with the two teams who had received prior byes.