Although hepatitis B serology screening has been recommended for HIV care, it has not been routinely performed. We aimed to assess compliance and timing of hepatitis B serology screening among HIV-infected patients in a resource-limited setting. A cross-sectional study was conducted in Thailand. Compliance, timing of hepatitis B serology screening, and factors associated with no HBsAg screening were determined. A total of 416 HIV-infected patients with 61% males were enrolled. Median (range) age at HIV diagnosis was 34 (16-75) years and 92% had heterosexual risk. Proportion of HBsAg screening and prevalence of positive HBsAg were 69.2% and 9.0%, respectively. There was no difference in the proportion of no HBsAg screening during the period 1990-2008 (p = 0.865). Proportion of anti-HBs and anti-HBc screening were 40.9% and 21.2%, respectively. HBsAg was screened before or on the day of anti-HIV testing in 9.1% and before antiretroviral therapy (ART) initiation in 27.2%. By Kaplan-Meier analysis, median time from anti-HIV testing to HBsAg screening was 55.9 (95% confidence interval [CI] 43.9, 68.3) months. By multivariate logistic regression, duration of HIV infection (odds ratio [OR] 1.14; 95% CI 1.07, 1.21), no anti-HBs screening (OR 1.65; 95% CI 1.4-2.63), and no anti-HCV screening (OR 2.60; 95% CI 1.62, 4.17) were associated with no HBsAg screening before ART initiation. In conclusion, compliance with hepatitis B serology screening was relatively low and late. Educational program regarding hepatitis B serology screening, identification of barriers, and interventions to eliminate these barriers in resource-limited settings are crucial to improve HIV care.