Canine distemper is a highly contagious viral disease in carnivores and represents a serious threat for both wild and domestic animals. The aim of our study was to monitor the occurrence of the canine distemper virus in wildlife from the Czech Republic, reveal the H gene heterogeneity in positive samples and perform subsequent phylogenetic analysis. In total, 412 wild animals of 10 species were included in the study: 219 red foxes (Vulpes vulpes), 79 European badgers (Meles meles), 47 European otters (Lutra lutra), 40 stone martens (Martes foina), 10 pine martens (M. martes), 7 raccoons (Procyon lotor), 5 undetermined martens (Martes sp.), 2 wolves (Canis lupus), 1 European polecat (Mustela putorius), 1 free-ranging ferret (Mustela putorius furo), and 1 free-ranging American mink (Neovison vison). Most animals were found dead or were killed by hunters during hunting seasons in the years 2012–2020 and came from all 14 regions of the Czech Republic. In the animals that were hunted, symptoms such as apathy, loss of shyness or disorientation were reported. Canine distemper virus (CDV) was detected by real-time RT-PCR in the tissues of 74 (18%) of the animals, including 62 (28%) red foxes, 4 (10%) stone martens, 3 (43%) raccoons, 2 (20%) pine martens, 2 (2.5%) European badgers and 1 (20%) undetermined marten. There was a statistical difference in positivity among animal species (p < 0.0001), regions (p = 0.0057), and the years of sampling (p = 0.0005). To determine the genetic characteristics of circulating variants of CDV in wildlife, 23 of 74 CDV variants were partially sequenced. Phylogenetic analysis showed that 21 variants belonged to the European lineage and two strains belonged to the European-Wildlife lineage. This study provides the first comprehensive overview of the prevalence and spatial distribution of CDV in wildlife in the Czech Republic, including molecular phylogenetic analysis of currently circulating CDV lineages.