The prevalence and genetic diversity of hepatitis C infection in women attending antenatal clinics in two regions of England was investigated to inform future surveillance and control measures. Women booking into antenatal care are routinely offered a test for immunity to rubella. Serum residues from these tests were unlinked, anonymized and archived as part of the Unlinked Anonymous Prevalence Monitoring Programme (UAPMP). The serum specimens were tested for anti-HCV using a cost-effective pooling strategy. After taking into account differential sampling from the UAPMP serum archive, the adjusted overall prevalence of anti-HCV was 0.43% (95% CI: 0.32-0.53) in London and 0.21% (95% CI: 0.14-0.28) in the Northern and Yorkshire region. Restriction fragment length polymorphism of amplified HCV RNA identified type 3a as the most common HCV genotype in these antenatal women. The prevalence of anti-HCV in antenatal women in the UK is low and consistent with that expected from injecting drug use.