In crop breeding program, information about genetic dissimilarity on breeding resources is very important to corroborate genealogical relationships and to predict the most heterozygotic hybrid combinations and inbred breeding. This study aimed to evaluate the genetic variation in Kenyan sunflower breeding lines based on simple sequence repeat (SSR). A total of 83 alleles were detected at 32 SSR loci. The allele number per locus ranged from 2 to 7 with an average of 2.7 alleles per locus detected from the 24 sunflower accessions and the average value of polymorphic information contents (PIC) were 0.384. A cluster analysis based on the genetic similarity coefficients was conducted and the 24 sunflower breeding resources were classified into three groups. The principal coordinates (PCoA) revealed 34% and 13.38% respectively, and 47.38% of total variation. It was found that the genetic diversity within the Kenyan sunflower breeding resources was narrower than that in other sunflower germplasm resources, suggesting the importance and feasibility of introducing elite genotypes from different origins for selection of breeding lines with broader genetic base in Kenyan sunflower breeding program.