A new 3.8-kb allele at the 5' region of the PGK 1 locus detected by the probe pSPT/PGK is reported. This variant was discovered in the Cabecar and Guaymi, two Chibchan Amerindian groups of Costa Rica. So far, a polymorphism that consists of an EcoRI/BglI (1.3-kb) variable site within an EcoRI/BglII (1.7-kb) fragment when DNA is simultaneously digested with EcoRI, BglI and BglII is known to occur in black and Caucasian populations. These two alleles were also found in the Amerindians tested. The newly described band is due to the lack of the BglII site situated 1.7 kb downstream from the EcoRI site and to the cleavage of another BglII site 2.1 kb downstream from the lacking one. This variant might be restricted to some Amerindian groups and perhaps also to Asiatic populations. Thus, it could be a useful marker in evolutive studies and for forensic applications. Moreover, the presence of a third allele in populations with Amerindian ancestry can increase the heterozygosity of the region disclosed by the pSPT/PGK probe, thus improving its application in issues dealing with X-chromosome activation ratios in females.