Sorghum (Sorghum bicolor (L.) Moench) is the most stable and important food security crop in Ethiopia accounting for nearly 40% of human calorie intake. Knowledge of the natural genetic composition of the crop provides the option to further exploit its genetic potential through breeding. However, there are limited reports on the genetic variability of Ethiopian sorghum using a medium-throughput marker system. Hence, the current study was designed to evaluate the genetic variability of released and landrace Ethiopian sorghum genotypes using polymorphic microsatellite markers. A 92 sorghum accessions collected from five Ethiopian ecological zones were targeted using 12 SSR markers. The study resulted in 77 alleles across the entire loci and populations. All the used microsatellite loci were highly polymorphic with PIC ranging from 0.66 to 0.82 and an overall mean of 0.76. The analysis confirmed the presence of high gene diversity ranging from 0.71 to 0.84 with overall mean of 0.79. There was a higher genetic differentiation (FST=0.21) showing the presence of moderate gene flow. The analyzed molecular variances indicated the existence of large genetic differentiation (FST=0.21) where 90% of the total variation was accounted for within populations genetic variability, leaving only 10% for the among populations variation. The PCoA, clustering, and population structure did not cluster the studied populations into a separate groups according to their geographical areas of sampling due to the presence of considerable gene flow (Nm= 2.13). In conclusion, based on the overall evaluated loci the highest intra-population diversity was observed among populations of North Gondar (Het= 0.75) and South Tigray (Het= 0.74), and hence these areas can be considered as hot spots for the identification of genotypes for breeding program. Therefore, the present study generated valuable information for sorghum breeding programs, and for conservation measures.