Pathologic plasma lipoprotein cholesterol levels play a key role in the development and pathogenesis of human atherosclerotic cardiovascular diseases. Plasma cholesterol homeostasis is regulated by genetic predispositions and environmental factors. Animal models showing aberrant plasma cholesterol levels are used for the identification and analysis of novel causative genes. Here, we searched for inherited hypocholesterolemia phenotypes in randomly mutant mice which may contribute to the detection of disease protective alleles. In the Munich ENU mouse mutagenesis project, clinical chemistry blood analysis was carried out on more than 15,500 G1 offspring and 230 G3 pedigrees of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to a decreased plasma total cholesterol level. We identified 66 animals consistently showing hypocholesterolemia. Transmission of the altered phenotype to the subsequent generations led to the successful establishment of 14 independent hypocholesterolemic lines. Line-specific differences were detected by clinical chemistry analysis of plasma HDL cholesterol, LDL cholesterol and triglycerides. Thus, we successfully established a novel panel of ENU-derived mutant mouse lines for their use in the identification of alleles selectively influencing the plasma cholesterol homeostasis. Such findings may be subsequently used for humans and other species.