Open AccessThe AFLP and SSR markers were used to estimate the genetic diversity of 25 Brassica napus hybrids attending Guizhou regional test. The fingerprints obtained with both the AFLP and SSR markers revealed high levels of heterozygosity. Nine AFLP primer combinations produced 16 loci, while 11 SSR primer pairs generated 22 loci. The mean of expected heterozygosity, Shannon’s information index, and genetic differentiation obtained by SSR were higher than those by AFLP, indicating that the SSR methodology evaluated genetic diversity among B. napus more efficiently than the AFLP approach. The higher level of genetic diversity detected by SSR markers was contributed to the lower genetic similarity estimates based on SSR markers (mean 0.69) as compared to AFLP markers (mean 0.73). While the AFLP technique was suitable for identification and DNA fingerprinting of B. napus germplasm. Based on AFLP and SSR analysis, it was concluded that B. napus hybrids had high level genetic diversity.