Coffee is an established plant for its flavor and has high commercial use. In Nepal, the popularity of coffee is increasing for its high economic value. However, its diversity and the status of its genetic mapping have not been studied in Nepal. In the present study, the genetic diversity of 28 coffee accessions was assessed by using twenty-four SSR markers with the aim of studying the variation of coffee in accord with the genetic markers from a molecular approach. With the use of DNA extraction and marker selection for its amplification using PCR tools, a total of 81 loci from SSR were identified. Of all SSR 63.22% showed for mean polymorphism. The mean polymorphic information content of SSR was 0.38, which showed low genetic diversity of SSR markers among Coffea genotypes. On the basis of the SSR marker, the unweighted pair group method with arithmetic mean (UPGMA) dendrogram constructed showed a similar group of distribution among 28 accessions, which was further supported by a principle coordinate analysis scatter plot. The phylogenetic relationships among the accessions were assessed by SSR marker, which also showed low diversity in coffee genotypes. Our study demonstrated the use of SSR markers in diversity analysis as the data were informative and highly reproducible for evaluating relationships among coffee cultivars in Nepal. The use of more markers systems and a high genotype pool would have been beneficial in accessing more accurately. Regardless, the information from the phylogenetic relationship study could be useful for breeding, varietal improvement, and for conservation programs.