Clear cell renal cell carcinoma (ccRCC) is one of the most lethal urological malignancies with high tumor heterogeneity, and reliable biomarkers are still needed for its diagnosis and prognosis. WEE family kinases function as key regulators of the G2/M transition, have essential roles in maintaining cellular genomic stability and have the potential to be promising therapeutic targets in various tumors. However, the roles of WEE family kinases in ccRCC remain undetermined. In the present study, we first explored multiple public datasets and found that PKMYT1 was up-regulated in both RCC tumors and cell lines. Expression levels of PKMYT1 were highly associated with pathological stage and grade. Kaplan-Meier curves showed that high PKMYT1 expression was associated with lower overall survival and disease-free survival. Receiver operating characteristic curves revealed that the expression of PKMYT1 could better distinguish ccRCC from normal samples. Functional enrichment analysis demonstrated that cell cycle- related pathways and epithelial to mesenchymal transition (EMT) might be potential mechanisms of PKMYT1 in ccRCC tumorigenesis. Moreover, knockdown of PKMYT1 in vitro attenuated the proliferation, migration and invasion of RCC cell lines, promoted cell apoptosis and prevented the EMT phenotype in vitro. In conclusion, our study demonstrated that PKMYT1 has the potential to act as a diagnostic and prognostic biomarker for RCC patients. Targeting PKMYT1 may be considered as a new potential therapeutic method and direction in RCCs.