The present study aims to determine the level of malatox resistance in peach fruit fly (Bactrocera zonata).The surface film petri dish technique and molecular markers such as Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeats (ISSR) were used to make fingerprinting. Adult flies collected from different areas in Egypt; Siwa Oasis, Assiut, Ismailia, Kafr-Elsheikh and Giza were compared to the laboratory strain. The laboratory strain was tested with the organophosphorus insecticide (malatox) with surface film petri dish technique to estimate the concentration mortality lines. An approximate LC₉₉ value was selected to discriminate for resistance in five field colony populations. Data revealed that individuals collected from Siwa fields exhibited the highest resistance value to malatox followed descendengly by individuals of Assiut, Ismailia, Kafr-Elsheikh and Giza, respectively. RAPD analysis of malatox resistant of B. zonata flies showed considerable differences, the five primers detected a total of 176 bands, with an average of 35.2 bands per primer and percentage of polymorphism ranged from 66% (P13) to 100% (C16). Similarly, ISSR marker gave 100% polymorphism among the five studied primers, produced 161bands with an average of 32.2 bands per primer and found to be potential markers for resistant.These results suggested that malatox treatment created genetic alterations in field B. zonata flies and that may be a reason to initiate or create resistant strains from this dangerous insect pest.