In the United States, men who have sex with men (MSM) constitute the risk group in which the prevalence of new HIV infection is increasing. The percentage of undiagnosed HIV infection and HIV risk behaviors in MSM and non-MSM participating in an emergency department-based rapid HIV screening program were compared. Medical records of all male patients participating in the program from May 2008 to October 2010 were reviewed. MSM were identified as male or male-to-female patients reporting oral and/or anal sex with a male. Males eligible for testing were aged 18 or older, English-speaking, not known to be HIV infected, and able to decline testing. A total of 6672 males were approached for testing; 5610 (84.1%) accepted, 366 (6.5%) were MSM, and 5244 (93.5%) were non-MSM. A total of 90.7% were black. Median age was 41. Fifty-nine MSM (16.1%) were diagnosed with HIV compared to 81 (1.5%) non-MSM. MSM were 10 times more likely than non-MSM to have undiagnosed HIV infection (odds ratio [OR] 10.4, 95% confidence interval [CI] 7.3, 14.0). HIV-infected MSM (median age, 26) were younger than non-MSM (median age, 41). HIV-infected non-MSM were 2 times more likely than MSM to have CD4 counts less than 200 cells per microliter. MSM were more likely to report previous HIV testing (OR 1.9, 95% CI 1.4, 2.5) and risk behaviors, including sex without a condom (OR 2.0, 95% CI 1.5, 2.6), sex with an HIV-infected partner (OR 14.6, 95% CI 8.3, 25.6) and sex with a known injection drug user (OR 4.1, 95% CI 2.0, 8.4). Further investigation of emergency department-based HIV testing and risk reduction programs targeting MSM is warranted.