We analysed the characteristics of the pregnancies with a previously undetected HIV infection in a national observational study of pregnant women with HIV in Italy. In a total of 443 pregnancies with available date of HIV diagnosis, 118 were characterized by a previously undetected HIV infection (26·6%, 95% CI 22·5–30·8). The following factors were independently associated with this occurrence in a multivariate analysis (adjusted odds ratios; 95% CIs): foreign nationality (5·1, 2·8–9·3); no pre-conception counselling (35·9, 4·8–266·1); first pregnancy (2·1, 1·2–4·0); asymptomatic status (6·8, 1·5–30·6). Women with previously undetected infection started antiretroviral treatment significantly later during pregnancy (P<0·001). Missed diagnosis was responsible for one case of transmission. A high rate of previously undetected HIV infection was observed. This suggests a good HIV detection during pregnancy, but also the need to reinforce HIV testing strategies among women of childbearing age. We identified some determinants which may be considered for intervention measures.