The identification of predictive DNA markers for pork quality would allow U.S. pork producers and breeders to more quickly and efficiently select genetically superior animals for production of consistent, high quality meat. Genome scans have identified QTL for tenderness on pig chromosome 2 which have been fine-mapped to the calpastatin locus. The objectives of this study were to identify the sequence variation in calpastatin that likely affects tenderness in commercial-level pig populations and to develop definitive DNA markers that are predictive of pork tenderness for use in marker assisted selection programs. We resequenced the calpastatin regulatory and transcribed regions in pigs with divergently extreme shear force values in order to identify possible mutations that could affect tenderness. One hundred ninety-four single nucleotide polymorphisms (SNP) were identified in this sequence and thirty-one SNPs were found in predicted transcription factor binding sites. We tested 131 polymorphisms in our research population and a subset (40) of these in samples of industry pigs for association with objective measures of tenderness. We identified four SNPs that were consistently associated with pork tenderness in all of the populations studied, representing 2,826 pigs from four distinct populations. Gel shift assays were designed for these SNPs and 12 other polymorphic sites. Six sites demonstrated a gel shift when probes were incubated with nuclear extract from muscle, heart or testis. Four of these sites, a Sp1 site around nucleotides 12978 and 12979, a potential Tef site at nucleotide 25587, an unknown site at nucleotide 48699, and Mef-2/TATA sites with SNPs at positions 49223 and 49228 were allele-specific in binding nuclear proteins. The allele frequencies for the tender alleles were similar (0.11-0.36) in the four different commercial populations. These four SNPs were not in complete linkage disequilibrium with each other and may independently affect calpastatin expression and/or tenderness. These markers should be predictive of pork tenderness in industry populations.