Grasscutter (Thryonomys swinderianus) is a hystricomorpha rodent that inhabits mainly Sub-Saharan Africa and its meat is highly relished by most people in this region. Attempts are being made to domesticate it to forstall the negative impact of its hunting on the environment. DNA was extracted from liver and lung of one male animal and sequenced using three Roche454 genome shotgun approaches which generated a total of 704,466 reads with an average length of 377 bp. A total of 95,802 sequences contained microsatellites out of which 949 sequences were selected for primer design. One hundred and fifty five primer pairs were synthesized and tested, out of which 28 di-repeats and 10 tri-repeats were found to be polymorphic. The Mendelian inheritance of all polymorphic markers was determined using a grasscutter family of six individuals. A total of 89 grasscutters from three agro-ecological zones in Ghana namely, Guinea Savannah (n = 20), Forest (n = 15), Coastal Savannah (wild, n = 22; domestic, n = 32), were genotyped with the 10 tri-repeat markers. Nei’s genetic distance indicated that the two Savannah populations are closer than the Forest population. The markers developed in this study are useful for kinship analysis and individual identification.