Ascosphaera apis (Maassen ex Claussen) Olive et Spiltoir is a causal agent of chalkbrood disease and is widely found in mainly honeybee colonies worldwide. Chalkbrood disease rarely causes colony death, but severe infections can lead to a decrease in honeybee populations and as a result, a decrease in honey production or beekeeping byproducts. Therefore, it is important to determine the distribution and prevalence of this disease agent in a certain region in terms of controlling of it. In this study, it was aimed to identify chalkbrood disease agents affecting honeybee colonies especially in beekeeping places in Turkey, to isolate and identify them using morphological and molecular methods, especially ITS gene sequencing. Morphologically, pure cultures of clinically suspected samples in terms of chalkbrood disease were created and microscopically evaluated. After that, genomic DNA isolation was performed from pure cultures to amplify ITS gene region and the resulting gene sequences were used in molecular identification and phylogenetic analysis. For this purpose, a total of 1.193 hives were randomly selected from 400 apiaries in 40 provinces throughout Turkey between 2018 and 2019. As a result, the presence of A. apis was detected in 7 hives in 2018 and 19 hives in 2019, as a result a total of 26 samples were found to be positive. Accordingly, the infection rate was determined as 2.18%. The obtained results are thought to be important in terms of determining the spread rate of A. apis and the rate of chalkbrood disease in Turkey.