There are no data concerning the genotypic analysis of hepatitis B virus (HBV) infection in Brazilian dialysis centers. Serum samples from all hemodialysis patients (n = 282) in Goiânia City, Central Brazil, were tested for hepatitis B surface antigen (HBsAg). An overall prevalence of 12.0% was found, ranging from 0 to 33.3% depending on dialysis centers. Positive samples (n = 34) were submitted to serological subtyping by monoclonal ELISA and HBV DNA detection by polymerase chain reaction (PCR). Among 30 PCR-positive samples, 26 were genotyped by use of the line probe assay technology (INNO-LiPA HBV, Innogenetics, Gent, Belgium). HBV genotypes A (50. 0%) and D (46.2%) were the most frequently found whereas genotype F (3.8%) was rare in this population. Serological subtypes adw2 (44. 1%) and ayw3 (41.2%) were dominant. By contrast, adw4 and ayw2 were found at a low frequency (2.9%). A correlation was observed in the distribution of genotypes and subtypes by dialysis center. Genotype D and subtype ayw3 were predominant in 2 hemodialysis centers whereas genotype A and subtype adw2 were predominant in the others. The findings of high HBsAg prevalence rates restricted to certain dialysis centers and the data obtained through genotyping and serological subtyping suggest HBV nosocomial transmission in these hemodialysis centers.