Paspalum dilatatum is an excellent warm-season forage grass and soil conservation plant in subtropical regions. However, information on P. dilatatum cultivation in southwestern China is limited. A preliminary evaluation of the genetic diversity and adaptability of six P. dilatatum germplasms was conducted in Guizhou based on inter simple sequence repeat (ISSR) markers and field trials. Fifteen polymorphic primers were screened from 50 ISSR primers. A total of 141 bands were generated, 124 of which were polymorphic, yielding 88.63% polymorphic bands. The number of polymorphic loci detected for the different germplasms ranged from 23 to 61, and the percentage of polymorphic loci was 40.67%. The mean number of alleles was 1.23, and the effective allele number was 1.15. Nei's genetic diversity index was 0.08. The Shannon information index was 0.12, and the genetic similarity coefficient (GSC) ranged from 0.6002 to 0.8955, indicating a relatively rich genetic base among the germplasms. The unweighted pair group method with arithmetic mean approach was used to construct the clustering map. Using a GSC of 0.83 as the threshold value, the six germplasm materials were divided into four clusters, indicating that ISSR molecular markers can be effectively applied to analyze the genetic diversity of P. dilatatum germplasms. Cluster analysis of 12 growth characteristics of the germplasms of P. dilatatum showed that germplasm 4 exhibited strong tillering ability and a high crude protein content. Germplasm 3 showed high plant height, fresh yield and hay yield. Germplasm 1 showed the lowest forage yield and crude protein content. Germplasms 2, 5 and 6 showed excellent crude protein and crude fat contents; the remaining indicators showed intermediate performance. This study provides important references for the utilization and variety selection of introduced P. dilatatum resources in the karst areas of Guizhou.