Next Generation Sequencing technology has become an efficient approach for analyzing functional genomics and facilitates discovery of molecular biomarkers for various biological applications. This study is aim to discover all simple sequence repeat (SSR) biomarker candidates and associated genetic functions from two different tilapia strains. The two transcriptome datasets, Nile Tilapia and Mozambique Tilapia, were retrieved from the public SRA database for transcriptome assembly and functional biomarker discovery. We adopted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases for gene function annotations. A total of 116,781 SSRs and 126,324 SSRs were identified from these two tilapia strains, and there were 31,950 SSR variations could be detected within annotated genes. To illustrate effective and efficient performance of our proposed system, we used the keywords of “skeletal system development” as an example for discovering associated SSR markers. There are three genes annotated with function of skeletal system development, and which possess 5 different SSRs within the gene sequences. Under such a particular functional constraint, a gene cluster can be automatically defined and all linked polymorphic SSRs could be identified between the two different species. These identify linked SSR markers are important for further analysis on genetic regulation, genetic disease detection, gene therapy, or cultivation of various agricultural products.