An alkylation of 2-oxo(thioxo)-thieno[2,3-d]pyrimidine-4-ones is a versatile and feasible route to biologically active pyrimidines. Experimental and theoretical approaches via different types of atomic partial charges at different semiempirical and DFT levels of theory were used for the investigation of the regioselectivity of alkylation of 2-oxo(thioxo)-thieno[2,3-d]pyrimidine-4-ones. Alkylation of ambident 2-oxo(thioxo)-thieno[2,3-d]pyrimidine-4-one anion with substituted allyl (methallyl/cinnamyl) chlorides selectively leads to the formation of N- or S-substituted pyrimidines which is totally confirmed via NMR and XRD studies. Partial atomic charges are considered for prediction of a most reactive atom of the ambident anionic pyrimidine moiety in the three solvents (ethanol, N,N-dimethylformamide, dimethyl sulfoxide) modeled via CPCM and COSMO. It was shown that thioxo analog selectively reacts upon exocyclic sulfur atom in the solvents with different polarity, whereas in the case of dioxo pyrimidine anion tends to undergo N-alkylation.