Objectives: This study aimed to determine the prevalence of blood transfusiontransmitted infections (TTIs), among blood donors in Pakistan, specifically HIV, hepatitis B virus (HBV), hepatitis C virus (HCV), syphilis, and malaria. Methods: Data records of all registered blood donors (n = 120 968) during 2008– 2019, at a blood transfusion center in a tertiary care hospital were assessed. Frequency of the seropositive donors for HIV, HCV, HBV, syphilis, and malaria was analyzed. Results: The overall age range of the donors was 25–65 years. Nearly all were male (99.0%). HCV, syphilis, and malaria were more prevalent among those aged 26–35 years. Most donors (81.1%) were residents of Islamabad city. The infection with the highest prevalence among the screened blood donors was HCV (1.5%; 95% CI: 0.423– 0.661) followed by syphilis (0.8%; 95% CI: 1.149–1.432). HCV and syphilis were most frequently observed in blood group B positive (B+) donors while HIV was more common in those who were O+. The frequency of co-infection of syphilis with HCV and HIV was 0.02% and 0.01%, respectively. Conclusions: Among male blood donors, the most prevalent TTI infection was HCV followed by HIV; the latter is on the rise. HCV and syphilis are the most frequent co-infections.