Abstract Microsatellites are small and are codominant markers that can be amplified with polymerase chain reaction. Both prokaryotic and eukaryotic organisms possess large amounts of the microsatellites repeat. Many microsatellites have high mutation rates that generate the high levels of allelic diversity necessary for genetic studies of processes acting on ecological time scales. The high variability of microsatellites provided the foundation for their successful application in a wide range of fundamental and applied fields of biology. However, de novo isolation is needed for most species hence in this study we tried to mine the microsatellite marker using PCR-based isolation of microsatellite arrays (PIMA) on Blue spotted mudskipper, Boleophthalmus boddarti a fish uniquely restricted to coastal and estuarine habitat was also commercially important. Out of three trials, seven microsatellite repeats were detected but only three repeat types (AAG)₄, (TCAG)₃ and (CT)₄ can be useful as microsatellite marker following PHOBOS V3.3.12 analysis. Meanwhile, the detection of octa (AATACAT)₂, penta (TGACA)₂ and heptanucleotides (GGAGATA)2 were unable to be continued as functional microsatellite marker as there were missense variants and interruptions detected either on forward or reverse strand of the repeat. Thus, PIMA method could be considered as tedious and detected low yields of microsatellite markers. Nevertheless, the conventional method for generating microsatellite markers from PCR based methods could be done with in silico mining of microsatellite sequences from DNA sequence databases or next generation sequencing (NGS).