Host-seeking ticks were collected in the Northern Apennines, Italy, by dragging at 35 sites, at altitudes ranging from 680 and 1670 m above sea level (asl), from April to November, in 2010 and 2011. Ixodes ricinus (4431 larvae, 597 nymphs and 12 adults) and Haemaphysalis punctata (11,209 larvae, 313 nymphs, and 25 adults) were the most abundant species, followed by Haemaphysalis sulcata (20 larvae, five nymphs, and 13 adults), Dermacentor marginatus (42 larvae and two adults) and Ixodes hexagonus (one nymph). Greatest numbers of ticks were collected at locations characterised by southern exposure and limestone substratum, at altitudes <1400 m asl; I. ricinus was most abundant in Turkey oak (Quercus cerris) wood, whereas H. punctata was mostly collected in hop hornbeam (Ostrya carpinifolia) wood and on exposed rocks. Ixodes ricinus was also found up to 1670 m asl, in high stand beech (Fagus sylvatica) wood. The overall prevalence of Borrelia burgdorferi sensu lato (sl) in 294 host-seeking I. ricinus nymphs was 8.5 %. Borrelia garinii was the most frequently identified genospecies (64.0 % of positive nymphs), followed by B. valaisiana, B. burgdorferi sensu stricto, B. afzelii, and B. lusitaniae. Based upon the comparison with the results of previous studies at the same location, these research findings suggest the recent invasion of the study area by the tick vector and the agents of Lyme borreliosis.