An investigation was carried out on the distribution and biodiversity of steinernematid and heterorhabdtid entomopathogenic nematodes (EPN) in nine regions of Italy in the period 1990–2010. More than 2000 samples were collected from 580 localities and 133 of them yielded EPN specimens. A mapping of EPN distribution in Italy showed 133 indigenous EPN strains belonging to 12 species: 43 isolates of Heterorhabditis bacteriophora, 1 of H. downesi, 1 of H. megidis, 51 of Steinernema feltiae, 12 of S. affine, 4 of S. kraussei, 8 of S. apuliae, 5 of S. ichnusae, 3 of S. carpocapsae, 1 of S. vulcanicum, 3 of Steinernema ‘isolate S.sp.MY7’ of ‘S. intermedium group’ and 1 of S.arenarium. Steinernematids are more widespread than heterorhabditids and S. feltiae and H. bacteriophora are the most commonly encountered species. Sampling sites were grouped into 11 habitats: uncultivated land, orchard, field, sea coast, pinewood, broadleaf wood, grasslands, river and lake borders, caves, salt pan and moist zones; the soil texture of each site was defined and the preferences of habitat and soil texture of each species was assessed. Except for the two dominant species, S. feltiae and H. bacteriophora, EPN occurrence tends to be correlated with a specific vegetation habitat. Steinernema kraussei, H. downesi and H. megidis were collected only in Sicily and three of the species recently described – S. apuliae, S. ichnusae and S. vulcanicum – are known only from Italy and seem to be endemic.