The diversity and prevalence of cancer disease is a chocking fact. Accordingly, the need for new, effective and safe anticancer agents is now mandatory. Fourteen novel tricyclic and tetracyclic benzothienopyrano and benzothienopyranopyrimidine derivatives 2, 3a-d, 4a-c, 5-8 and 9a,b were synthesized. Compounds 3b and 3d have been chosen by the NCI-USA for the 60-cell lines One-Dose screening and Five-dose assay. Both 3b and 3d exhibited very potent cytotoxic effects (GI50 2.0 M) against almost all cell lines. 3b showed TGI values ranging from 0.463–4.75 M while 3d showed values of 0.571–6.28 M. The best sensitivity patterns for both 3b and 3d were against MDA-MB-435 subpanel (melanoma cancer) while they possessed a null sensitivity towards all leukemic cancer subpanels.