Travel influences the spread of non-B subtypes of HIV in Europe. The aim of this study was to assess whether the spread of non-B subtypes occurred in Croatia, a country with a low level HIV infection. Phylogenetic analysis of the reverse transcriptase and protease genes was undertaken in 145 Croatian patients with the following risks for HIV-infection: men who have sex with men (n = 64), heterosexual contact (n = 54), intravenous drug use (n = 21), bisexual contact (n = 4) and intravenous drug use plus heterosexual exposure (n = 2). The sample included 25 seamen and 13 steady female sexual partners of seamen. Overall, 38 (26%) of HIV-patients were infected with non-B subtypes. Non-B subtype infections were found only in patients with a possible heterosexual exposure. Non-B infections were most frequent in seamen and their regular female partners (in 27 of 38 seamen and their partners, 71%). The relative distribution of non-B subtypes was as follows: A, 8%; C, 26%; F, 3%; CRF01_AE, 8%; CRF02_AG, 34%; CRF05_DF, 5%; CRF06_cpx, 3%; CRF10_CD 8%; and subtype U, 5%. There were altogether 19 heterosexual couples, phylogenetic analysis confirmed that the paired samples were related in all cases. Non-B subtypes were found only in individuals with a heterosexual risk, mainly in seamen and their partners.