Sugar and acidity levels are the main criteria of general fruit quality and for citrus juices pulp, in particular. The constituents of the acidity (organic acids) and the sweetness (glucose, fructose and sucrose) and the genes involved in their regulation have seldom been used to explore Citrus genetic diversity. We evaluated the juice composition of primary metabolic components for 87 varieties belonging to the eight major Citrus species grown under the same environmental and cultivation conditions by HPLC. We investigated the sequence polymorphism of nine candidate genes encoding for key enzymes of sugars and organic acids metabolic pathways by single strand conformation polymorphism (SSCP). Whatever the biochemical or molecular analyses, the observed structure of Citrus diversity was organized around three groups corresponding to the ancestral species (mandarin, pummelo and citron). As expected, the secondary species were closely related to their putative ancestors except for Citrus aurantium. Biochemical diversity was strongly correlated to molecular SSCP diversity at the genus level but not at the intraspecific level. Compared with other molecular marker types, higher diversity has been observed with SSCP technology, which makes it suitable for future quantitative trait loci mapping approach on gene polymorphism in citrus pulp acidity and sweetness regulation.