Non-syndromic tooth agenesis (ns-TA) is one of the most common dental anomalies characterized by the congenital absence of at least one permanent tooth (excluding third molars). Regarding the essential role of genetic factors in ns-TA aetiology, the present study aimed to identify novel pathogenic variants underlying hypodontia and oligodontia. In a group of 65 ns-TA patients and 127 healthy individuals from the genetically homogenous Polish population, the coding sequences of 423 candidate genes were screened using targeted next-generation sequencing. Pathogenic and likely pathogenic variants were identified in 37 (56.92%) patients, including eight nucleotide alternations of genes not previously implicated in ns-TA (, , , , , and ). However, since only single variants were detected, future research is required to confirm and fully understand their role in the aetiology of ns-TA. Additionally, our results support the importance of already known ns-TA candidate genes (, , , , , , , and ) and provide additional evidence that ns-TA might be an oligogenic condition involving the cumulative effect of rare variants in two or more distinct genes.