Major League Soccer and the United Soccer Leagues jointly unveiled the A-League and D3 Pro League affiliates for the 2001 MLS and USL Seasons. Recent USL realignment and membership changes have led to several alterations within the affiliate system, most notably the addition of the A-League’s expansion Portland Timbers as an affiliate for the 2000 MLS Cup Champion Kansas City Wizards, the San Jose Earthquakes and the TFC Champions Cup winner Los Angeles Galaxy. One other new A-League club, the Charlotte Eagles, formerly of the D3 Pro-League, will also change affiliates as it joins alliances with D.C. United and the Miami Fusion after serving as the Tampa Bay Mutiny’s affiliate since 1999. An updated list of affiliates for the 2001 MLS Season:
| MLS | A-League | D3 Pro League |
| Chicago Fire | Indiana Blast | Western Mass Pioneers |
| Milwaukee Rampage | ||
| Minnesota Thunder | ||
| Pittsburgh Riverhounds | ||
| Colorado Rapids | Cincinnati Riverhawks | Northern Nevada Aces |
| Nashville Metros | Utah Blitzz | |
| Seattle Sounders | ||
| Columbus Crew | Cincinnati Riverhawks | Reading Rage |
| Indiana Blast | ||
| Pittsburgh Riverhounds | ||
| Rochester Raging Rhinos | ||
| Dallas Burn | Atlanta Silverbacks | Tucson Fireballs |
| El Paso Patriots | ||
| Milwaukee Rampage | ||
| Nashville Metros | ||
| D.C. United | Charlotte Eagles | Hampton Roads Mariners |
| Hershey Wildcats | Northern Virginia Royals | |
| Richmond Kickers | ||
| Kansas City Wizards | Atlanta Silverbacks | Greenville Lions |
| Portland Timbers | ||
| Rochester Raging Rhinos | ||
| Los Angeles Galaxy | El Paso Patriots | Arizona Sahuaros |
| Portland Timbers | ||
| San Diego Flash | ||
| Seattle Sounders | ||
| Miami Fusion | Charlotte Eagles | Wilmington Hammerheads |
| Charleston Battery | ||
| Atlanta Silverbacks | ||
| New England Revolution | Connecticut Wolves | Boston Bulldogs |
| Hershey Wildcats | New Hampshire Phantoms | |
| Long Island Rough Riders | Rhode Island Stingrays | |
| MetroStars | Connecticut Wolves | New Jersey Stallions |
| Long Island Rough Riders | South Jersey Barons | |
| Hershey Wildcats | ||
| San Jose Earthquakes | El Paso Patriots | Stanislaus United Cruisers |
| San Diego Flash | Bay Area Seals | |
| Seattle Sounders | Chico Rooks | |
| Portland Timbers | ||
| Tampa Bay Mutiny | Charleston Battery | Carolina Dynamo |
| Minnesota Thunder | ||
| Richmond Kickers |