The occurrence of colistin resistance has increased rapidly amongEnterobacteralesaround the world. We performed a national survey of plasmid-mediated colistin resistance in human clinical isolates through a retrospective analysis of samples from 2009 to 2017 and a prospective sampling in 2018–2020. The aim of this study was to identify and characterize isolates withmcrgenes from various regions of the Czech Republic using whole genome sequencing (WGS). Of all 1932 colistin-resistant isolates analyzed, 73 (3.8%) were positive formcrgenes. Most isolates carriedmcr-1(48/73) and were identified asEscherichia coli(n= 44) andKlebsiella pneumoniae(n= 4) of various sequence types (ST). Twenty-five isolates, includingEnterobacterspp. (n= 24) andCitrobacter freundii(n= 1) carrying themcr-9gene were detected; three of them (Enterobacter kobeiST54) co-harbored themcr-4andmcr-9genes. Multi-drug resistance phenotype was a common feature ofmcrisolates and 14% (10/73) isolates also co-harbored clinically important beta-lactamases, including two isolates with carbapenemases KPC-2 and OXA-48. Phylogenetic analysis ofE. coliST744, the dominant genotype in this study, with the global collection showed Czech isolates belonged to two major clades, one containing isolates from Europe, while the second composed of isolates from diverse geographical areas. Themcr-1gene was carried by IncX4 (34/73, 47%), IncHI2/ST4 (6/73, 8%) and IncI2 (8/73, 11%) plasmid groups. Small plasmids belonging to the ColE10 group were associated withmcr-4in three isolates, whilemcr-9was carried by IncHI2/ST1 plasmids (4/73, 5%) or the chromosome (18/73, 25%). We showed an overall low level of occurrence ofmcrgenes in colistin-resistant bacteria from human clinical samples in the Czech Republic.