OBJECTIVES: To estimate the prevalence of HIV, hepatitis C virus (HCV) and syphilis in injecting drug users (IDUs) in Russia. METHODS: Unlinked anonymous cross-sectional survey of 1473 IDUs recruited from non-treatment settings in Moscow, Volgograd and Barnaul (Siberia), with oral fluid sample collection for HIV, HCV antibody (anti-HIV, anti-HCV) and syphilis testing. RESULTS: Prevalence of antibody to HIV was 14% in Moscow, 3% in Volgograd and 9% in Barnaul. HCV prevalence was 67% in Moscow, 70% in Volgograd and 54% in Barnaul. Prevalence of positive syphilis serology was 8% in Moscow, 20% in Volgograd and 6% in Barnaul. Half of those HIV positive and a third of those HCV positive were unaware of their positive status. Common risk factors associated with HIV and HCV infection across the cities included both direct and indirect sharing of injecting equipment and injection of home-produced drugs. Among environmental risk factors, we found increased odds of anti-HIV associated with being in prison in Moscow, and some association between official registration as a drug user and anti-HIV and anti-HCV. No associations were found between sexual risk behaviours and anti-HIV in any city. CONCLUSIONS: HIV prevalence among IDUs was markedly higher than city routine surveillance data suggests and at potentially critical levels in terms of HIV prevention in two cities. HCV prevalence was high in all cities. Syphilis prevalence highlights the potential for sexual risk and sexual HIV transmission. Despite large-scale testing programmes, knowledge of positive status was poor. The scaling-up of harm reduction for IDUs in Russia, including sexual risk reduction, is an urgent priority.