Both the haplotype distribution and the mutational spectrum of the phenylalanine hydroxylase (PAH) gene has been defined for the Chilean phenylketonuria (PKU) population. Mutation analysis was performed using a combined approach of screening for common European and Oriental mutations and application of the DGGE scanning method in the remaining uncharacterized alleles. A total of 16 different mutations have been identified, including two novel ones, Q232X and IVS11nt5. The most frequent mutations were IVS10nt-11 and V388M present both in the 13% of the mutant chromosomes. The rest of the mutations are rare. The haplotype association including VNTR and STR alleles, was examined to investigated the origin and distribution of PAH alleles in Chile. Our results are consistent with Southern Europeans as the major source of PAH mutations in Latin America. However, we have also detected mutations from East and Central Europe, such IVS12nt1, R408W and R252W. It is clear that the PKU mutation present in each Latin American country varies with the demographic profile and specific mutation scanning is necessary in each population both for diagnostic and prognostic purposes. The correlation between the genotypes and the phenotypes is consistent with the emerging pattern of mutation severity deduced from previous studies in related populations.