The objectives of this study is to characterize HIV-serology-discordant couples diagnosed at a referral hospital in Thailand and to identify risk factors for HIV transmission among married couples. Firstly, cross-sectional analysis was conducted from July 2000 to October 2002. Out of 216 HIV-positive married men who knew the HIV status of their wives, the median number of sexual contacts in 63 men with HIV-negative wives was 6 times per month before the disclosure of HIV status, which did not differ from 153 men with HIV-positive wives. The majority of men with HIV-negative wives never used condoms. The median duration of marriage was 7 years for both groups. Unlike in previous reports, men with HIV-negative wives were significantly more symptomatic (P < 0.01), and their CD4+ counts and viral loads did not differ from men with HIV-positive wives. Secondarily, 71 initially discordant couples were longitudinally followed until March 2005. Four were seroconverted out of 132.24 person-years of observation. In multivariate analysis incorporating sex, age, CD4+ count and sexual contact without a condom, shorter duration of marriage (<2 years) was found to be the only risk factor significantly associated with HIV transmission (hazard ratio of 15.2, P = 0.04). Individuals substantially exposed to HIV but remaining HIV-negative are accumulated in discordant couples identified in a hospital, except in recently married couples.