Isoenzymatic polymorphism analysis was used to determine genetic variability among species and hybrids of Citrus spp. and one accession of Poncirus trifoliata (L.) Raf. Ten enzymatic systems aspartate aminotransferase (AAT), acid phosphatase (ACP), leucine aminopeptidase (LAP), 6-phosphogluconate dehydrogenase (6-PGD), isocitrate dehydrogenase (IDH), phosphoglucoisomerase (PGI), phosphoglucomutase (PGM), diaphorase (DIA), shikimate dehydrogenase (SKD) and peroxidase (PRX) were analyzed. Twenty loci and 48 alleles were identified. Sweet orange cultivars (C. sinensis (L). Osbeck) showed the highest polymorphism with the largest number of heterozygous loci, although the alleles of those loci were the same in all cultivars, with the exception of Westin and Lima graúda. Mandarins (C. reticulata Blanco) exhibited diverse patterns, whereas Poncirus trifoliata (L.) Raf. showed high variability with all Citrus species and hybrids. Exclusive phenotypes were observed in some enzymatic systems, and similar patterns were found among interspecific hybrids and their putative parents.