The objective of the present study was to gain a better understanding of the epidemiology of encephalomyocarditis virus (EMCV) infections in pigs by applying molecular techniques. The diagnostic potential of a reverse transcription-PCR (RT-PCR) targeting 286 nucleotides at the 3' end of the gene which encodes the viral polymerase was assessed with experimental and field samples. In addition, the use of the amplified sequences for an epidemiological study was evaluated. The heart was clearly shown to be the most suitable organ. The detection limit was determined to be 1 viral particle in 100 mg of heart tissue. The sensitivity and specificity of the assay on the basis of the results obtained in this study were 94 and 100%, respectively. Phylogenetic analysis of the amplified sequences classified EMCVs in two distinct lineages. Group A consists of the reference strain ATCC 129B, all isolates collected between 1991 and 1994 in Belgium in association with reproductive failure, and all Greek isolates. All Belgian isolates collected since the first isolation of EMCV in relation to myocardial failure in fatteners in Belgium group together with the isolates from Cyprus (1996 and 1997), Italy (1986 to 1996), and France (1995) in group B irrespective of their pathogenicity. The analyzed part of the 3D gene differed by 13.0% between Groups A and B. In contrast to the sequence homogeneity of the Belgian isolates collected between 1991 and 1994, molecular diversity, which ranged between 0 and 2%, was observed among the Belgian isolates collected in 1995 and 1996. Among all Greek isolates the diversity ranged between 1 and 8%. However, this diversity does not seem to reflect geographical links between the outbreaks. A RT-PCR for the rapid and specific diagnosis of EMCV in a variety of clinical samples followed by nucleotide sequence analysis proved to be valuable for molecular epidemiological studies.