To investigate the karyotype characteristics and evolutionary relationships of different populations of Psammochloa villosa, we studied the karyotype of six populations of P. villosa in the Inner Mongolia Plateau using the traditional squashing method. The result showed that: (1) the chromosome number of six populations of P. villosa was constant, all of which were 2n = 2x = 46. (2) There was a total of four chromosome types including the center centromere type (M), centromeric subtype (m), subcentral centromere type (sm) and proximal centromeric type (st), and the amount of centromeric subtype is the largest in all populations. (3) The karyotype formulas were different among six populations of P. villosa. (4) There were four karyotype types of P. villosa, including 1A, 2A, 1B and 2B. The average arm ratio of chromosomes was 1.29-1.62, the length ratio was 1.73-2.68. (5) The karyotype asymmetry coefficient was 55.96%—59.95%, which implied the karyotype of P. villosa was symmetrical and primitive. More importantly, the karyotype asymmetry coefficient of 37 population of P. villosa was the largest among all the populations, and the evolution degree of it was much higher than that of others, whereas that of 34 population of P. villosa was the smallest, and the evolution degree was much lower than that of others. (6) The six populations of P. villosa were clustered into two categories. Among them, the 37 population of P. villosa was singly clustered into one group, while other populations were clustered into one group, and they had the relatively distant relationship. In the present study, we reported the karyotype characteristics and evolutionary relationship of different populations of P. villosa for the first time, which could provide the cytological evidence for systematic evolution and selection of fine germplasm resources of P. villosa in the future.