Methylmalonic acidemia (MMA)-affected patients may have developmental, hematological, neurological, metabolic, ophthalmological, and dermatological clinically abnormal findings. This study aimed to identify mutations in 13 Chinese MMA cases. We provided genetic counseling, treatment, and prenatal diagnosis for the families with MMA. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed and the results were confirmed by gas chromatography and mass spectrometry (GC/MS). Variant screening in probands was performed by targeted next-generation sequencing. Identified variants were confirmed by Sanger sequencing. Of these 13 MMA cases, seven were isolated MMA, and among them, six were caused by variants in MMUT and one was caused by a variant in MCEE. The other six cases were MMA with homocystinuria, which was caused by variants in MMACHC. We found six novel variants in three MMA-causing genes as follows: c.2008G>A, c.301_302insTA, c.984delC, and c.319A>T of MMUT; c.445T>C of MMACHC; and c.296T>C of MCEE. We provided prenatal diagnosis for two families with MMA at their next pregnancy, and one family had a healthy newborn. In conclusion, our findings expand the spectrum of genotypes in MMA. Effective genetic counseling is required to allow awareness of the patients' families that MMA disease is treatable and a good prognosis can be obtained.