Background: Chikungunya virus (CHIKV), responsible for a debilitating febrile illness, has seen an increase in prevalence among Pakistani population in recent years. Though mainly transmitted through bite of Aedes mosquitoes, the other transmission route of particular concern is transfusion-related, especially during the outbreak season when asymptomatic individuals are capable of transmitting the virus through this route. Therefore, we designed this study to know the infectious and immune status of healthy blood donors against CHIKV. Material and Methods: This was a cross-sectional study conducted on healthy blood donors at the Blood bank of Aga Khan University from 1st July 2018 to 28th February 2019. The sampling method employed was consecutive sampling. Three hundred and sixty healthy blood donors were screened for the presence of CHIKV IgM antibodies and nucleic acid using ELISA method and RT-PCR, respectively.Results: Of the 360 blood donors screened, 1.1% (n=4) tested positive for CHIKV IgM however, none were found to be viremic at the time of blood donation. The mean age of seropositive donors was 32.5 years (IQR: 37.5 – 28) and all were residents of Karachi city. CHIKV seropositivity was significantly associated with residence in the central district of Karachi (p=0.02).Conclusion: This screening study is suggestive of potential subclinical infection to CHIKV among healthy blood donors and provides evidence for routine screening of this virus to eliminate risks of transfusion related transmission