Cardiolipin (CL) and its precursor phosphatidylglycerol (PG) are important anionic phospholipids widely distributed throughout all domains of life. They have key roles in several cellular processes by shaping membranes and modulating the activity of the proteins inserted into those membranes. They are synthesized by two main pathways, the so-called eukaryotic pathway, exclusively found in mitochondria, and the prokaryotic pathway, present in most bacteria and archaea. In the prokaryotic pathway, the first and the third reactions are catalyzed by phosphatidylglycerol phosphate synthase (Pgps) belonging to the transferase family and cardiolipin synthase (Cls) belonging to the hydrolase family, while in the eukaryotic pathway, those same reactions are catalyzed by unrelated homonymous enzymes: Pgps of the hydrolase family and Cls of the transferase family. Because of the enzymatic arrangement found in both pathways, it seems that the eukaryotic pathway evolved by convergence to the prokaryotic pathway. However, since mitochondria evolved from a bacterial endosymbiont, it would suggest that the eukaryotic pathway arose from the prokaryotic pathway. In this review, it is proposed that the eukaryote pathway evolved directly from a prokaryotic pathway by the neofunctionalization of the bacterial enzymes. Moreover, after the eukaryotic radiation, this pathway was reshaped by horizontal gene transfers or subsequent endosymbiotic processes.