| Microsatellites are one option for characterizing cattle populations; that information can be used as a basis for the development and maintenance of local cattle. This study aims to evaluate genetic diversity and relationships using microsatellite markers between the populations of Gorontalo cattle in Gorontalo Province, Indonesia, using 3 microsatellite markers. A total of 126 samples of local bovine blood were collected, which consisted of North Gorontalo (n=28), Gorontalo (n=52), Bonebolango (n=37), PO (n=3), and Bali (n=6), were used in this study. The Genomic DNA Mini Kits were used for DNA extraction for an analysis fragment in the microsatellite DNA region using ILSTS017, HEL13, and BM1818 primers. A total of 74 alleles were identified across entire populations. The expected heterozygosity ranged from 0.407±0.216 (Bali) to 0.716±0.050 (Gorontalo), and the observed heterozygosity ranged from 0.471±0.084 (Bonebolango) to 0.778±0.222 (PO). F statistical analysis includes FIS 0.038, FIT 0.248, and FST 0.231. The three microsatellite markers were moderate (0.25-0.5) to highly informative (PIC>0.5). The research showed that Bali cattle were distinct from all other cattle populations, while Gorontalo-Bonebolango admixture and North Gorontalo cattle were mixed with the PO cattle population. In conclusion, markers used were highly informative and polymorphic in investigating genetic diversity in Bonebolango, Gorontalo, and North Gorontalo populations, while the genetic relationship among cattle populations was divided into two main clusters, i.e., the Bali and PO populations, which closely reflect the breeding process in the research area. This information will be useful for future development and maintenance of local cattle in Gorontalo Province, Indonesia.