Manipulation of mouse genome has merged as one of the most important approaches for studying gene function and establishing the disease model because of the high homology between human genome and mouse genome. In this study, the chemical mutagen ethylnitrosourea (ENU) was employed for inducing germ cell mutations in male C57BL/6J mice. The first generation (G1) of the backcross of these mutated mice, totally 3172, was screened for abnormal phenotypes on gross morphology, behavior, learning and memory, auditory brainstem response (ABR), electrocardiogram (ECG), electroretinogram (ERG), flash-visual evoked potential (F-VEP), bone mineral density, and blood sugar level. 595 mice have been identified with specific dominant abnormalities. Fur color changes, eye defects and hearing loss occurred at the highest frequency. Abnormalities related to metabolism alteration are least frequent. Interestingly, eye defects displayed significant left-right asymmetry and sex preference. Sex preference is also observed in mice with abnormal bone mineral density. Among 104 G1 generation mutant mice examined for inheritability, 14 of them have been confirmed for passing abnormal phenotypes to their progenies. However, we did not observe behavior abnormalities of G1 mice to be inheritable, suggesting multi-gene control for these complicated functions in mice. In conclusion, the generation of these mutants paves the way for understanding molecular and cellular mechanisms of these abnormal phenotypes, and accelerates the cloning of disease-related genes.