In continuation to our research program concerned with structural modification of thieno[2,3-d] pyrimidines with the purpose of enhancing their anticancer activity, a series of S-alkyl thieno[2,3-d] pyrimidinone was designed and synthesized to investigate the effect of varying the linker and aliphatic or aromatic amine on the anticancer activity. The structure of the synthesized compounds has been elucidated on the basis of elemental analyses and spectroscopic methods (IR, 1H-NMR, 13C-NMR and MS). The in vitro cytotoxic activity of the newly synthesized compounds was evaluated against two human cell lines: prostate cancer (PC-3) and colon cancer (HCT-116). Compounds 4a, 4b, 11b and 11c exhibited potent anticancer activity against PC-3 cell line while 11b and 11c displayed promising anticancer activity against HCT-116. Compound 4a exhibited 5.78 fold more potent activity against PC-3 while compound 11b displayed 3.62 fold higher activity against HCT-116 compared to Imatinib.