Slovakia is one of the European countries reporting a low prevalence of human immunodeficiency virus 1 (HIV-1) infection. The HIV-1 subtype B was previously the most prevalent in Slovakia and mostly associated with men having sex with men (MSM) (Habekova et al., 2010). The aim of this study was to determine the distribution of individual HIV-1 subtypes among the virus-positive individuals diagnosed in 2009-2012 in Slovakia and to compare these data with those from 2004-2008. The HIV-1 subtype B exhibited the highest prevalence (86.1%) and was mostly associated with MSM as stated before. HIV-1 non-B subtypes were found in 13.9% of patients, led by CRF01_AE (6.3%) and followed by subtype C (2.8%), F1 (1.4%), CRF03_AB (1.4%), G (0.7%), CRF02_AG (0.7%), and CRF12_BF (0.7%). The incidence of non-B subtypes varied in 2009-2012, showing a maximum in 2012. MSM represented the main risk group for total HIV-1 (68.1%) as well as B subtype (63.9%) infections in Slovakia. In contrast, the heterosexual contact represented the main route of HIV-1 acquisition of the non-B subtype virus infection. Together with the increase in the number of patients infected with the viruses of non-B subtype also other changes were observed: infections with the viruses of non-B subtype became more common among MSM and not so strongly associated with heterosexual contact. All these findings may help to design the right strategy for retaining of HIV epidemic in Slovakia at low level.