Clinical assays for the primary evaluation of congenital hemoglobin (Hb) disorders must detect and identify a variety of Hb variants. We analyzed hemolysates containing Hb variants with similar charge to evaluate the diagnostic sensitivity and specificity of automated capillary isoelectric focusing (CIEF). Peak separation was observed for each variant in samples containing Hb S, D, and G. The calculated isoelectric points (pl) of these variants were significantly different such that each could he identified in a single run with pi as the sole criterion of identification. The pi of Hb C was significantly different from that of Hb E, C-Harlem, and O-Arab. Hb E, C-Harlem, and O-Arab had similar pi and were not readily differentiated. Hb Koln, M-Saskatoon, Aida, and S/Aida hybrid were readily separated from common Hb variants and detected by CIEF. We conclude that CIEF exhibits both diagnostic sensitivity and specificity, and that pi is an objective and specific criterion of Hb variant identification.