Cholesterol gallstone formation is a response to interactions between multiple genes and environmental stimuli. To determine the subset of cholesterol gallstone susceptibility (Lith) genes possessed by strains CAST/Ei (susceptible) and 129S1/SvImJ (resistant), we conducted quantitative trait locus (QTL) analyses of an intercross between these strains. Parental strains and F(1) mice of both genders were evaluated for gallstone formation after consumption of a lithogenic diet for 8 wk. Gallstone susceptibility of strain CAST was predominantly due to cholesterol hypersecretion. Male intercross offspring were genotyped and phenotyped for cholesterol gallstone formation after consumption of the lithogenic diet for 10 wk. Linkage analysis was performed using PSEUDOMARKER software. One significant, new QTL was detected and named Lith13 [chromosome (Chr) 5, 30 cM]. Statistical analyses and QTL fine mapping suggest this QTL may comprise two closely linked loci. We confirmed the presence of Lith6 (Chr 6). Suggestive QTL were detected on Chrs 1, 2, 5, 14, and 16. The QTL on Chrs 2 and 16 confirmed previously identified, suggestive QTL. Therefore, they were named Lith12 (101 cM) and Lith14 (42 cM), respectively. We identified candidate genes based on known function and location and performed mRNA expression analyses using both parental strains and intercross progeny for preliminary evaluation of their contributions to gallstone formation. Cebpb (Lith12), Pparg (Lith6), and Slc21a1 (Lith6) displayed expression differences. Our work continues to demonstrate the genetic complexity and to elucidate the pathophysiology of cholesterol gallstone formation. It should facilitate the development of new approaches for treating this common human disorder.