Hb Acharnes or [β53(D4) Ala - Thr] is a newly discovered unstable hemoglobin variant. It has been reported in very few literatures across the world and no cases have been reported from India till date. Hb Acharnes is known to interact with β°-thalassemia to produce thalassemia intermedia and heterozygotes may present with borderline HbA2 levels. Here we report a rare case discovered during routine screening of thalassemia and hemoglobinopathies in a 19 year old pregnant lady. To emphasize the diagnostic difficulties and importance of detection of a rare hemoglobin variant Hb Acharnes [β53(D4) Ala - Thr] in an asymptomatic patient in West Bengal, India. The 19 year old asymptomatic pregnant lady P1+0, LMP - 21.01.2011 reported in antenatal OPD of Burdwan Medical College & Hospital, Burdwan, West Bengal, India for routine follow up. After proper antenatal check up her blood was collected as a routine screening for Thalassemia, EDTA blood was taken on 5th April 2011 and was subjected to Hemoglobin estimation by Cyanmethemoglobin method, Cell parameters in automated cell counter (SYSMEX KX21) and Hemoglobin analysis by HPLC in BIORAD VARIANT system. The patient was normal with respect to clinical examination and urinalysis. Routine blood counts revealed mild microcytic hypochromic anemia and on HPLC an unknown band (retention time 2.2 minutes, 21.2%, appearing as a shoulder of HbA0 band) of hemoglobin variant was discovered with normal level of HbF and HbA2. Hemoglobin analysis of her mother showed similar pattern while her father and her husband had normal Hb-HPLC pattern. The unknown hemoglobin variant was identified as Hemoglobin Acharnes or [β53 (D4) Ala - Thr] by the Biorad laboratories upon consulting the standard chromatogram patterns for this particular hemoglobin variant. Only Haemoglobin Electrophoresis by conventional gel technique may miss the case and the HPLC pattern may be used as a standard control for identification.