Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a widespread genetically inherited enzyme disorder caused by mutation in the G6PD gene located in X chromosome. Although sequencing of the gene is considered as the standard method for detection of mutations, but the process is expensive, laborious, time-consuming and not possible to perform in low resource settings. Thus, the present work was aimed to identify the G6PD variants using Polymerase Chain Reaction – Restriction Fragment Length Polymorphism (PCR-RFLP) based method among the ethnic groups of malaria endemic Northeastern part of India. STANDARD G6PD Analyzer was utilized for screening to identify the G6PD deficient malarial patients. Molecular analysis of the deficient samples was done using PCR-RFLP technique. A total of 17 variants were explored using different restriction endonucleases. Five variants viz., Orissa (63.8 %), Kalyan-Kerala/Jamnagar/Rohini (9.02 %), Mediterranean (8.3 %), A+ (11.8 %) and Mahidol (1.38 %) were detected among the patients. G6PD Orissa was the major variant among the study population. In malaria endemic regions like Northeast India, a mass screening program for identification of G6PD variants needs to be adopted.