Expression and genetic variants of the thyroid hormone responsive protein (THRSP) gene has been reported to be associated with marbling and fatty acid composition in cattle. The study was undertaken to investigate the effect of 2 single-nucleotide polymorphisms (SNP) in the THRSP gene, namely c.88 G>A and c.194 C>T, on morphological and biochemical traits of the longissimus muscle. Genotyping of 247 F₂-generation bulls of a Charolais × Holstein cross revealed that both SNPs were in complete linkage disequilibrium in our data set with 37 bulls of the GG/CC, 110 of the AG/CT, and 100 of the AA/TT diplotype. Muscle fiber composition, intramuscular fat cell and marbling traits were analyzed using image analysis. Fatty acid concentrations/proportions were determined with gas chromatography using internal standard procedure as fatty acid methyl esters. The F₂ design yielded substantially increased variation within the population compared with both founder breeds regarding muscle structure, fat cell, and marbling data within F₂ bulls ranged from 4.5 standard deviations (cross sectional area of intermediate muscle fibers) to 7.4 standard deviations (area percentage of slow muscle fibers). Furthermore, absolute and relative fatty acid composition exceeded variation of muscle structural traits with differences between individuals up to 11.3 standard deviations (C21) indicating extreme divergence in fat deposition within the population. However, association analysis indicated no clear effect of THRSP-SNP diplotype on longissimus muscle structure and intramuscular fat deposition, but showed an indication on association of the THRSP-SNPs with muscle fatty acid composition (P < 0.05).