Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy. This deficiency in erythrocytes has a prevalence of 0.51 +/- 0.109 in the Caucasoid male population of Portugal. The frequency for deficiency-conferring genes is 0.39% in the Portuguese population. In the herein study populations males from areas of Portugal presenting with the highest prevalence of G6PD deficiency (Castelo Branco, Setúbal, Faro, and Lisbon) as well as similar subjects located in the border Center/North area of the country (Viseu) have been analyzed for biochemical parameters and screened for mutations and haplotype-associated mutations commensurate with G6PD deficiency. Six intragenic restriction fragment length polymorphisms (RFLPs) were studied: exon 5, nt 376 A -->G, FokI; intron 5, nt 611 C--> G, PvuII; intron 8, nt 163 C--> T, BspHI; exon 10, nt 116 G --> A, PstI; exon 11, nt 1311 C--> T, BclI; and intron 11, nt 93 T -->C, NlaIII. New haplotypes were constructed with the inclusion of intron 11, nt 93 T--> C, NlaIII, and only 5 of 64 possible haplotypes were found to show a marked linkage disequilibrium for several RFLPs and also for mutations and specific haplotypes. The control population (n = 168 males) presented the G6PD B variant and corresponded to haplotypes I (- - + + - -), Ia (- - + + - +), and VIIa (- - + + + +), in 91.8, 2.3, and 5.9%, respectively. The PCR and sequencing analysis of extracted DNAs from the deficient G6PD group showed 48.6% (16/33) of individuals with the G6PD A- mutation, corresponding to haplotype VIa (+ + - + - +); 9% (3/33) with the Betica mutation and 18% (6/33) with the Santa Maria mutation, both of them associated with haplotype IVa (+ - - + \- +); 6.1% (2/33) with the Mediterranean mutation associated with haplotype VIIa; 12.3% (4/33) with the Seattle mutation, 3.0% (1/33) with Gaohe mutation; and a new mutation, 3.0% (1/33), which we designated by G6PD Flores, all of them associated with haplotype I.