The objective of this article is to describe the prevalence of maternal Hepatitis C infection globally and by HIV serostatus. Unlinked anonymous testing on 474,539 samples from newborn babies was carried out, selected through stratified sampling by year and HIV status, in six Spanish Autonomous Regions, from 2003 to 2006. Results were 0.15% for global anti-hepatitis C virus (HCV), 29.69% and 0.10% for HIV(+) and HIV(-) women, respectively. Anti-HCV prevalence rate is low in these regions compared to Hospital cohorts. Although a high prevalence rate in HIV co-infected women is found, it tends to decrease along the period studied.