The HIV fusion inhibitor enfuvirtide (ENF/Fuzeon) targets the env gp41 transmembrane domain. Mutations in gp41 are associated with ENF resistance. We developed a prototype assay to genotype a 676-bp region spanning the heptad repeat domains (HR1 and HR2) of HIV-1 gp41. Plasma samples were collected from 126 HIV-1-infected blood donors in Cameroon, Brazil, Uganda, South Africa, Thailand, and Argentina. Based on analysis of gag p24, pol integrase, and env gp41 genes, the panel was composed of subtypes A/A2 (18), B (11), C (14), D (10), F/F2 (9), G (7), CRF01_AE (9), CRF02_AG (33), and recombinant strains (15). Genotyping was successful for 119 of the 126 samples (94.4%). Although numerous amino acid polymorphisms were detected in some samples, none had primary mutations associated with ENF resistance. The gp41 HIV-1 research reagents developed by Celera are useful tools for genotyping analysis of the gp41 region in diverse HIV-1 strains.