Abstract: In order to evaluate and compare the laboratory methods of separating and diagnosing of the mature honeybee`s contamination to mite varroa, we used four hives, which were contaminated by this kind of mite. All of these samples were taken during the early days of autumn when most of the mites lived on the mature honeybees. In each sample, 250 honeybees were collected in a glass container for separating mite from the mature honeybees. For this sake, we used ethanol 70%, ether, gasoline, sugar, heat, hot water and hot water together with detergent. In sugar shake method, we used both commercial sugar and sugar powder. All of these methods were experienced three times and each time on new samples. Results indicated that ethanol 70%, with separating power 95.41% ±1.54 and sugar powder, with separating power 89.82±1.69%, were the most effective methods in separating mite varroa from the mature honeybees. By using the statistical software SPSS -13.0 and χ2 test, the statistical comparison of these results indicated that there was no significant difference between ethanol and sugar powder shake method (p> 0.05). However, there was a significant difference between the ethanol roll method and other methods (p≤0.05). In addition, there was a significant difference between the sugar powder shake method and commercial using method (p≤0.05). Therefore, for the rapid and exact diagnose, we recommend to use ethanol 70% or sugar powder. Using sugar powder did not result to death of honeybee and had a high separating ability, no antivarroa drugs residue in hive, so it is advised for remedy of varroatosis.