We estimated the number of people unaware of their human immunodeficiency virus (HIV) infection in our province, Pavia (population 540 000) in Lombardy, Italy, by means of anonymous unlinked testing of 10 044 serum/plasma samples residual from clinical analyses at the outpatient clinic of Policlinico San Matteo in 2014 and 2015. Ethical and legal approval was obtained prior to study start. Samples were irreversibly anonymised, only retaining gender and 5-year age class. Five sample pools were tested for HIV using LIAISON® XL MUREX HIV Ab/Ag (DiaSorin, Saluggia, Italy). If the pool tested positive, individual samples underwent confirmatory tests, Innotest HIV Antigen mAb (Fujirebio Europe, Gent, Belgium) and HIV BLOT 2·2 (MP Diagnostics, Singapore). Among the 10 044 samples processed, eight were confirmed positive (0·08%, 95% confidence interval 0·03–0·16%), all were males and age was >50 in 3 (37·5%). If projected to the entire population of the Pavia province, this would result in approximately 1000 people unaware of their HIV infection, with age older than expected. In Italy, HIV testing is voluntary, universally free-of-charge and (upon request) anonymous. Nevertheless, this study demonstrates that it is suboptimally employed, and that new strategies and population-level actions will be needed to achieve better implementation of HIV testing and HIV control in our province.