We were asked to investigate a 54 year old male who was undergoing evaluation for sleep apnea. The patient had a low resting O2 saturation at 91%, but no signs of cardiac or pulmonary disease as indicated by a normal echocardiogram, normal spirometry, and normal high resolution CT of the chest. Arterial blood gases revealed a pO2 of 79 mm Hg on room air, but pCO2 was normal. The patient’s hemoglobin concentration was 13.7 g/dL. The possibility of a mutant hemoglobin was raised and HPLC revealed HbA2 = 2.6%, HbA = 51.4% and an unidentified mutant hemoglobin of 46%. Oxygen dissociation testing demonstrated a P50 of 39 mm Hg (normal 24–32) consistent with a low affinity hemoglobin. The isopropanol test showed that the hemoglobin variant was unstable. Trypsin digestion of the abnormal hemoglobin resulted in a smaller than normal HPLC peak for the β9 segment of β-globin, which is composed of amino acids 67 through 82, and the appearance of a new peak at 193 min. This peak did not match any of the known mutant hemoglobins contained in our reference bank. We PCR amplified and sequenced the β-globin gene. The patient was heterozygous for an A to C mutation in the second position of codon 66. This mutation changed the lysine to threonine and revealed that the hemoglobin variant was Hb Chico. This result was consistent with the loss of ~50% of the β9 peak in the trypsin digest due to loss of a cleavage site after amino acid 66, and the appearance of a new peak which was a hybrid segment of β8 and β9. This represents the first DNA sequence of hemoglobin Chico a mildly unstable hemoglobin variant with decreased oxygen affinity.