To determine the etiology of genital ulcers and to assess the prevalence of human immunodeficiency virus (HIV) infection in ulcer patients in 10 US cities, ulcer and serum specimens were collected from ~50 ulcer patients at a sexually transmitted disease clinic in each city. Ulcer specimens were tested using a multiplex polymerase chain reaction assay to detect Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus (HSV); sera were tested for antibody to HIV. H. ducreyi was detected in ulcer specimens from patients in Memphis (20% of specimens) and Chicago (12%). T. pallidum was detected in ulcer specimens from every city except Los Angeles (median, 9% of specimens; range, 0%-46%). HSV was detected in ≥50% of specimens from all cities except Memphis (42%). HIV seroprevalence in ulcer patients was 6% (range by city, 0%-18%). These data suggest that chancroid is prevalent in some US cities and that persons with genital ulcers should be a focus of HIV prevention activities.