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Background: Patients undergoing dialysis treatment and hemodialysis are at risk of viral infections due to inadequate cellular immunity. Objectives: The aim of the study was to determine the prevalence of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) in hemodialysis (HD) centers in North Cyprus. Methods: The present study reviewed the health records of 140 patients in two dialysis units that represented all HD units in North Cyprus. Serological markers for HBV, HCV, and HIV were determined by the immunoenzymatic assay using commercial diagnostic kits (Architect i2000 SR, Abbott, USA). HCV RNA, HBV DNA, and HIV RNA were determined quantitatively using polymerase chain reaction (PCR). Results: One hundred forty HD patients were included in the study, consisting of 39.3% (n = 55) female and 60.7% (n = 85) male patients. Five (3.6%) patients were anti-HCV positive, one (0.7%) patient was HBsAg positive, and one (0.7%) was anti-HIV positive. Anti-HCV and HBsAg were negative in all of the patients according to the PCR results. There were no significant differences between males (1.2%) and females (7.3%) in terms of anti-HCV positivity (P = 0.078), HBsAg seropositivity (P = 0.607), and anti-HIV seropositivity (P = 0.607). Conclusions: The prevalence of HBV, HCV, and HIV infection in hemodialysis patients in North Cyprus is moderate to low. The main reason for the significantly lower rates compared to other areas could be effective protective measures and national vaccination.