Olives infested with the olive fruit fly were collected by monitoring olive orchards in Canakkale area during 2006 and 2007, and in 2013 to study the level of organophosphate insecticide resistance in the acetyl cholinesterase gene. Single fly was used for genomic DNA isolation. All genomic DNA's were monitored by PCR-RFLP in the presence of resistance alleles. The assay was used to test the Glycine488Serine resistance mutation occurred in exon VI. The technique was established previously by mutagenesis due to introduced reverse primer that just reaches codon 488, creating Mbi I restriction site. The specimens screened in 2006 carried a resistance mutation (31.70%), and heterozygote mutation (65.21%), while only 3.10% of the specimens were organophosphate susceptible. The specimens screened in 2007carried a resistance mutation (54.14%), and heterozygote mutation (44.75%), while 1.10% of the specimens were susceptible. The specimens screened in 2013 carried a resistance mutation (81.77%) and heterozygote mutation (18.23%). There were no homozygous susceptible individuals found. This study presented the status and frequency of acetyl cholinesterase point mutation in the field collected olive flies against used organophosphate insecticides.