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Sequence-based genotyping was recently used to distinguish between the BVDV-1 and BVDV-2 species of the bovine viral diarrhoea virus (BVDV). Quite recently, a new putative species, BVDV-3, was also detected. The phylogenetic analysis of the 5′-untranslated region (UTR) and Npro region has revealed at least 17 distinct subtypes for BVDV-1 to date. The aim of this study was to further investigate the genetic heterogeneity of BVDV-1 in Italy, by analysing 173 virus sequences from isolates collected over an 18-year period (1997–2014). Viral RNA was extracted from the original biological samples identified as BVDV-1-positive. Reverse transcription (RT) and polymerase chain reaction (PCR) assays targeting a 288-base pair (bp) region of the 5′-UTR and a 428-bp region encoding the autoprotease Npro were performed, and the RT-PCR products were sequenced. The phylogenetic analysis of the 5 …