Siamese mud carp (Henichorynchus siamensis) is a freshwater teleost of high economic importance in the Mekong River Basin. However, genetic data relevant for delineating wild stocks for management purposes currently are limited for this species. Here, we used 454 pyrosequencing to generate a partial genome survey sequence (GSS) dataset to develop simple sequence repeat (SSR) markers from H. siamensis genomic DNA. Data generated included a total of 65,954 sequence reads with average length of 264 nucleotides, of which 2.79% contain SSR motifs. Based on GSS-BLASTx results, 10.5% of contigs and 8.1% singletons possessed significant similarity (E value < 10(-5)) with the majority matching well to reported fish sequences. KEGG analysis identified several metabolic pathways that provide insights into specific potential roles and functions of sequences involved in molecular processes in H. siamensis. Top protein domains detected included reverse transcriptase and the top putative functional transcript identified was an ORF2-encoded protein. One thousand eight hundred and thirty seven sequences containing SSR motifs were identified, of which 422 qualified for primer design and eight polymorphic loci have been tested with average observed and expected heterozygosity estimated at 0.75 and 0.83, respectively. Regardless of their relative levels of polymorphism and heterozygosity, microsatellite loci developed here are suitable for further population genetic studies in H. siamensis and may also be applicable to other related taxa.