Tooth agenesis is a high genetic heterogeneous disorder with more than 80 genes identified as associated molecular causes. The present study aimed to detect the possible pathogenic variants in a cohort of well-characterized probands with a clinical diagnosis of tooth agenesis. We performed whole-exome sequencing (WES) in 131 tooth agenesis patients with no previously identified molecular diagnosis. All the potential pathogenic variants were verified by Sanger sequencing in patients and their family members. Seventy-three patients were genetically diagnosed in 131 unrelated Chinese patients with tooth agenesis, providing a positive molecular diagnostic rate of 55.7%, including 53.8% (49/91) in the non-syndromic tooth agenesis (NSTA) group, and 60.0% (24/40) in syndromic tooth agenesis (STA) group. A total of 75 variants from 13 different genes were identified, including 33 novel variants, and WNT10A and EDA are the most common causative genes associated with non-syndromic and syndromic tooth agenesis, respectively. This study further extends the variant spectrum and clinical profiles of tooth agenesis, which has a positive significance for clinical practice, genetic diagnosis, prenatal counseling and future treatment.