Microsatellites or simple sequence repeats (SSRs) are short tandem repeats of DNA widespread in genomes and transcriptomes of diverse organisms and are used in various genetic studies. Few software programs that mine SSRs can be further used to mine polymorphic SSRs, and these programs have poor portability, have slow computational speed, are highly dependent on other programs, and have low marker development rates. In this study, we develop an algorithm named Simple Sequence Repeat Molecular Marker Developer (SSRMMD), which uses improved regular expressions to rapidly and exhaustively mine perfect SSR loci from any size of assembled sequence. To mine polymorphic SSRs, SSRMMD uses a novel three-stage method to assess the conservativeness of SSR flanking sequences and then uses the sliding window method to fragment each assembled sequence to assess its uniqueness. Furthermore, molecular biology assays support the polymorphic SSRs identified by SSRMMD. SSRMMD is implemented using the Perl programming language and can be downloaded from https://github.com/GouXiangJian/SSRMMD.