Human metapneumovirus (HMPV) is an important respiratory pathogen, especially among young children. The genetic characteristics of HMPV circulating in Croatia have not been studied so far. The aim of this study was to determine the incidence of HMPV infection in hospitalized children with acute respiratory tract infection (ARTI) in the season 2005/2006 in Croatia, as well as to perform the genotypic analysis of detected HMPV strains. From December 1 to March 31 nasopharyngeal secretions (NPSs) were collected from 402 inpatients up to 5 years of age with ARTI. NPSs were tested by real-time RT-PCR assay targeting the nucleoprotein (N) gene of HMPV. HMPV infection was detected in 33 patients (8.2%). To perform the phylogenetic study, partial nucleotide sequences were obtained for HMPV fusion (F) gene of 30 HMPV positive samples. Phylogenetic analysis showed the circulation of two main genetic lineages (A and B), with B lineages being prevalent. It also showed the existence of two sublineages within the group B (B1 and B2) and three subclusters within lineage A (A1, A2a and A2b). Further molecular analysis revealed point mutations in HMPV strains of sublineage B1. J. Med. Virol. 80: 1282–1287, 2008. © 2008 Wiley-Liss, Inc.