At least 16 couples were waiting at 7:15 a.m. inside the city's Moultrie courthouse, which houses the marriage bureau and is just blocks from the U.S. Capitol.
Sinjoyla Townsend, 41, and her partner of 12 years, Angelisa Young, 47, claimed the first spot in line just after 6 a.m.
"It's like waking up Christmas morning," Young said.
Washington will be the sixth place in the nation where gay marriages can take place. Because of a mandatory waiting period, however, couples won't actually be able to marry in the District of Columbia until March 9. Connecticut, Iowa, Massachusetts, New Hampshire and Vermont currently issue licenses to same-sex couples.
To deal with the expected crowd Wednesday, the marriage bureau will bring in temporary employees to help its regular staff, courthouse spokeswoman Leah Gurowitz said.
"Everybody who wants a marriage license is going to get one. It may take a little longer, but they will get their license," Gurowitz said.
To prepare, the marriage bureau has changed its license applications so they are gender-neutral, asking for the name of each "spouse" rather than the "bride" and "groom." And at civil marriage ceremonies to be performed in the courthouse, a booklet for the official performing the marriage now reads, "I now pronounce you legally married" instead of "I now pronounce you man and wife."
A marriage license application costs $35, and the marriage license $10. Couples who are already registered as domestic partners in the city can convert their registration into a marriage license by paying the $10 fee.
Supporters expected the day to be festive. A District of Columbia councilman who introduced the gay marriage bill planned to hand out boxes of vanilla and chocolate cupcakes to the first 200 couples in line.
Terrance Heath, 41, planned to be at the courthouse with his partner, Rick Imirowicz, 43. The two have been together for 10 years and have a 7-year-old and a 2-year-old, but Heath said Wednesday feels like "a step forward."
"My husband has always been my husband to me, but having that legal recognition, that legal protection, makes it easier to deal with any number of situations," said Heath, a writer and blogger. "If you tell people you're married, you don't really have to explain much beyond that."
The two, who live in Maryland, plan to marry on March 9, the first day possible.
The gay marriage law was introduced in the 13-member D.C. Council in October and had near-unanimous support from the beginning. The bill passed and D.C. Mayor Adrian M. Fenty signed it in December, but because Washington is a federal district, the law had to undergo a congressional review period that expired Tuesday.
Opponents, however, are still attempting to overturn the bill in court.