This study deals with the habitat preferences of medicinal leech Hirudo medicinalis Linnaeus, 1758 in the Czech Republic, in a highly agriculturally used and fragmented landscape, and provides the detailed information about the ventral coloration of this species. The study was conducted in 2010–2011 at 11 out of total 15 localities of H. medicinalis in the Czech Republic. At each locality a phytocenological sample of littoral and aquatic vegetation was recorded and physicochemical variables were measured. All captured leeches were measured and weighted, and a photo of ventral side was taken. Division of localities based on aquatic vegetation (TWINSPAN) formed groups of localities which corresponded to the population sizes of H. medicinalis. According to the abiotic factors localities with H. medicinalis were rich in carbon, poor in oxygen and situated in an area with warm climate. In this study four different ventral color patterns were described. During the ontogenetic development ventral side becomes darker, as indicated by the significant relationship between the percentage of dark color and leech size.