Microsatellites play an important role when investigating population and ecological genetics, although high effort in development and genotyping constitute a technical constraint and remains a major bottleneck. Here we use a microsatellite genotyping approach utilizing sequences of amplicons for allele calling (SSR-GBS) based on Illumina that requires less effort and time. The approach consist of development of highly polymorphic loci, sequencing of multiplexed PCR amplified microsatellites on an Illumina Miseq PE 300 platform and bioinformatic treatment of the sequenced data using custom scripts. The procedure allows automation in allele calling, which can be more reliably replicated and thereby removes biases that might prevent concatenation of datasets from different analyses. Additionally, the methodology enhances information content in the sequenced data beyond the traditional amplicon length (AL) approaches. Using 26 newly developed microsatellite markers and SSR-GBS we investigate the population genetic assessment of anthropogenically altered populations of East African Nile tilapia to show the potential of this genotyping approach. More precisely, we compare genotypic data generated considering AL and whole amplicon information (WAI). We found that genotypes based on WAI are not only able to recover a higher number of alleles but also a more detailed genetic structure pattern. We discuss the capability and importance of WAI allele calling and show perspectives for implementation in the future conservation genetic studies. More specifically, we demonstrate how the current markers and techniques might contribute useful information for studies concerning resources sustainable exploitation and conservation using the East African Nile tilapia.