Two previously identified single-nucleotide polymorphism markers located within the micromolar calcium-activated neutral protease gene (CAPN1) were evaluated for their association with variation in meat tenderness using one commercial sample of Simmental x Angus crossbred calves and one multibreed, crossbred research herd. The commercial sample included 362 animals sired by 23 registered Simmental bulls bred to unregistered Angus cows and represented current industry animals in which to test the predictive merit of the markers. The second sample was a research herd including 564 steers from the Germplasm Evaluation Cycle VII population at the U.S. Meat Animal Research Center, produced with semen from popular sires of the seven Bos taurus beef breeds with the most registrations in the United States (Angus, Charolais, Gelbvieh, Hereford, Limousin, Red Angus, and Simmental) on Angus, Hereford, and MARC III cows. These animals form a relatively outbred population that constituted a stringent test of the predictive merit of the genetic markers, although small groups were half-sibs. Warner-Bratzler shear force measurements were used to determine tenderness phenotypes for all animals. The populations were genotyped for two markers that predict variation at amino acid positions 316 and 530 of the mu-calpain polypeptide, produced by the CAPN1 gene. Minor allele frequencies for markers 316 and 530 in the commercial sample were 0.17 and 0.37, respectively, and in the Cycle VII animals, were 0.20 and 0.28, respectively. Both markers showed association with shear force in the commercial sample (P = 0.04) and the Cycle VII population (P = 0.02), supporting the hypothesis that they represent potential markers to aid selection for improved meat tenderness in commercial populations of beef cattle in the United States.