The present review provides a study on the structure features, reactions and synthetic methodologies of pyrido[4,3-d]pyrimidines. 5,6,7,8-Tetrahydropyrido[4,3-d]pyrimidine and related compounds have been used as starting materials for the multi-step synthesis of tetrahydropteroic acid derivatives. The aim of this review is to give an overview of the diverse methodologies that have been reported on the chemistry of pyrido[4,3-d]pyrimidines. The different synthetic routes have been grouped according to the way the pyrido[4,3-d]pyrimidine moiety has been created. Thus, the investigated compounds were obtained from pyridine and pyrimidine derivatives or from multicomponent synthesis. The mechanistic pathways of the reactions and the important biological applications are discussed.