Microsatellite is one of the most high-speed developing genetic markers for its wide application in molecular biology researches. It is proved to be a powerful marker-assisted tool in genetic relationship identification, the inheritance breeding, the population genetics, the physical map construction, the management and security of germplasm. These short tandem repeats loci are distributed throughout the eukaryotic genome. They represent not only highly conservative trait but also significant differentiation properties between individuals, making it advantageous over other molecular markers. Traditionally, hard labor is required for isolating these loci and the flanking sequences, including small fragment DNA library construction, DNA cloning, radioactive hybridization, sequencing, and microsatellite test. PIMA is a relatively simple microsatellite isolation technique which avoids not only library construction but also radioactivity manipulation. This approach builds on random amplified polymorphic DNA (RAPD) process but investigates microsatellite arrays by repeat-specific PCR rather than radioactive hybridization. PIMA screening microsatellites use one repeat-specific and two vector primers to run PCR. A number of useful vectors are widely circulated and the repeat-specific primer is easy to obtain. The advantages of obtaining both flank sequences simultaneously, no need of specific sequencing primers, the ease of operation, and well amplification of bacterial colonies persuade us of its high value. It prevails other tools because of its traits of cheaper, high-efficient, and relatively lower requirement of specialized equipment tool. Since no protocol is universal and perfect for every species, it is recommended that modification should be made according to the objective of the experiments. Existing examples serve as good sources of future works.