Background and ObjectivesVoluntary non‐remunerated blood donors (VNRBDs) are recognized as being crucial for the safety and sustainability of national blood supplies. Systems based on replacement donors (RDs) pose high risks of transfusion transmissible infections (TTIs). Currently, only 10%–13% of blood donations are voluntary in Pakistan. No large‐scale studies have been conducted to objectively evaluate the impact of the mode of donation on the frequency of TTIs, a gap this study aimed to fill.Materials and MethodsThe study was conducted at the Indus Hospital, Karachi. Data from a total of 591,820 blood donations were included from 1 October 2017 to 30 May 2021 and evaluated for type of donations and results of TTI testing, primarily performed on Architect i2000SR (Abbott). The TTIs tested include hepatitis B virus, hepatitis C virus, human immunodeficiency virus, syphilis and malaria.ResultsA total of 477,938 (80.7%) RDs and 113,882 (19.3%) VNRBDs were screened. Among these, 53,590 (9.06%) were positive for TTIs. There were 10.2% positive RDs (10.08–10.25 95% confidence interval [CI]) while 4.4% in VNRBDs (4.29–4.53 95% CI). Co‐infections were observed in 2367 (0.4%) RDs, while 159 (0.02%) in VNRBDs. Geographically, the highest frequency of TTIs was observed in semi‐urban areas of Sindh (11.2%) and Punjab (9.6%). A site‐wise comparison of TTIs in RD versus VNRBD showed significant differences (p‐value 0.00).ConclusionRDs are associated with higher frequencies of TTIs, compared with VNRBD. However, the study was unable to assess whether the significant difference was related to individual risk or repeat/first time status of the donors. Other important variables affecting frequency are the catchment area of the blood donors in Pakistan. Urban areas have less prevalence than semi‐urban areas.