Mitochondrial β-oxidation of long-chain fatty acids (LCFA) is essential for mammalian life. Because portions of this metabolic pathway are composed of enzymes that are coordinately regulated and share structural and functional similarities, we evaluated five of these enzyme genes for possible chromosomal linkages. Regulation of LCFA catabolism influences cell signal pathways and apoptosis, as well as energy production from LCFA. Partial cDNA fragments of the mouse mitochondrial proteins carnitine acetyltransferase (Crat), very-long-chain acyl coenzyme A dehydrogenase (Acadvl), the liver and muscle isoforms of carnitine acyltransferase I (Cpt1a and Cpt1b respectively), and a genomic PCR product of mitochondrial protein carnitine acyltransferase II (Cpt2) were used in a previously established mapping panel to determine their chromosomal locations. No pseudogenes were detected for any of the genes in Mus musculus, and all of the genes mapped to different chromosome locations, including the tissue-specific isoforms of carnitine palmitoyltransferase. Crat mapped to Chromosome (Chr) 2, at a position approximately 18 cM from the centromere and 2 cM proximal to the gene Ass1. Acadvl mapped to the middle of Chr 11, 8.3 cM distal to Il4 and 2.8 cM proximal to Mpmv2. Cpt1a mapped to the centromeric region of Chr 19, 8.7 cM proximal to Pomc-ps1. Cpt1b mapped to Chr 15, 4.9 distal to Gpt1 and 3.5 cM proximal to Wnt1. Cpt2 mapped to Chr 4 near the locus Pmv19.