Objective. To assess the level of haemoglobin A2 in HIV patients on antiretroviral therapy (ART) including zidovudine with untreated HIV patients. Material and Methods. The study was a case control study. A total of 185 patients were included in the study; the case group included 125 HIV patients who were on antiretroviral therapy (ART) including zidovudine and 60 were in the control group who were not receiving ART. The high-performance liquid chromatography (HPLC) was done and hemoglobin A2 level was observed; value more than 3.5% was considered significant. The Hb A2 percentages of HIV patients were compared with those of control using an unpaired t-test. Results. The mean of Hb A2 in case group was 3.462% (SD 0.675) and in control group it was 2.815% (SD0.246). The higher Hb A2 value was seen in significant number of treated patients than control group (P < 0.0001). Conclusion. The clinicians, pathologists, haematologists, and genetic counsellors should be aware of effects of nutritional anaemia and ART on Hb A2 to reduce the chances of misdiagnosis of β-thalassaemia especially in developing countries and for centres for antenatal screening.