To estimate HCV seroprevalence in subpopulations of women delivering live-born infants in the North Thames region in England in 2012, an unlinked anonymous (UA) cross-sectional survey of neonatal dried blood spot samples was conducted. Data were available from 31467 samples from live-born infants received by the North Thames screening laboratory. Thirty neonatal samples had HCV antibodies, corresponding to a maternal seroprevalence of 0·095% (95% confidence interval 0·067–0·136). Estimated HCV seroprevalences in women born in Eastern Europe, Southern Asia and the UK were 0·366%, 0·162% and 0·019%, respectively. For women born in Eastern Europe seroprevalence was highest in those aged around 27 years, while in women born in the UK and Asia-Pacific region, seroprevalence increased significantly with age. HCV seroprevalence in UK-born women whose infant's father was also UK-born was 0·016%. One of the 30 HCV-seropositive women was HIV-1 seropositive. Estimated HCV seroprevalence for women delivering live-born infants in North Thames in 2012 (0·095%) was significantly lower than that reported in an earlier UA survey in 1997–1998 (0·191%). Data indicate that the cohort of UK-born HCV-seropositive women is ageing and that, in this area of England, most perinatally HCV-exposed infants were born to women themselves born in Southern Asia or Eastern Europe.