Strawberry (Fragaria x ananassa Duchesne ex Rozier) is an economically important fruit crop grown commercially worldwide due to its known nutritional value. The demand for strawberries in the Philippines has been increasing but varietal development is slow due to the lack of genetic information on locally available germplasm. In addition, molecular marker resources for strawberries are limited. Here, we developed Simple Sequence Repeats (SSR) markers to characterize strawberries cultivated in tropical environments, particularly in the Philippines. In silico mining generated a total of 219,239 SSR markers, from which 160,025 unique markers were identified. Polymorphism scoring and the variation on the expected alleles resulting to the synthesis of the top 160 markers were validated using representative strawberry cultivars from the core collection. All 70 markers produced successful amplicons from all the genotypes, but only 67 markers displayed polymorphism. Using UPGMA, genetic similarities among the cultivars were calculated generating a dendrogram which categorized the strawberry cultivars into 3 groups. The grouping clearly showed the separation of the diploid Fragaria vesca from the octoploid F. x ananassa cultivars, considered as group 1. Furthermore, Groups 2 and 3 contained cultivars from different geographical origins (US, Japan, and North America). The eight novel markers developed and validated here can be added to the genetic marker resource for cultivar verification, assessment of genetic diversity, and most importantly, marker-assisted breeding.