Khat (Catha edulis (Vahl) Forssk.) is a herb from the Celastraceae family (also known as qat, gaad, or miraa). The leaves and stems are used medicinally and for recreational purposes. The communities that grow khat have identified different varieties based on perceived appearance and quality. This study aimed to evaluate the cytotoxicity of khat varieties grown in the Meru and Embu Counties of Kenya. Field studies were undertaken in the markets and farms in Meru and Embu Counties of Kenya to document and purchase local khat varieties. Dried khat was extracted with a 1:1 v/v MeOH: CH2Cl2 solvent and water. Cytotoxicity of extracts was determined in vitro by MTT assay against four normal and cancer cells namely; HeLa ATCC® CCL-2™, HCC1395 ATCC® CRL-2324™, Hep2 ATCC® CCL-23™, and Vero E6 ATCC® CRL-1586™. The khat varieties identified were Muti Mutiiri, Mugwanthingi, Gicheru, Karimi ka Nthiya, Muguka, Black colombo asili, Black mbaine, and white. The aqueous extracts of black colombo asili and black mbaine displayed the highest cytotoxic activity against HeLa cell lines having IC50 37.15 ± 1.75 µg/ml and 38.31 ± 2.05 µg/ml, respectively. Muguka and Muti Mutiiri varieties were not cytotoxic to the Vero E6 cell line with CC50 > 100 µg/ml. 75% (12/16) of extracts were cytotoxic to the Vero E6 cell line with CC50 ˂ 100 µg/ml. This study demonstrated that there is variability in activities between the identified khat varieties. Toxicity was observed in vitro due to observed cytotoxicity to the Vero E6 cell line.