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Termites of the genus Macrotermes (Termitidae: Macrotermitinae) are serious agricultural and structural pests, which also play vital roles in ecosystem functioning, and are crucial for the maintenance of tropical biodiversity. They are widely distributed, mainly in Southeast Asian countries; however, the parasitism of termites has been little researched. This research was conducted to identify and study the ecology of the parasitoids of termites at Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, Thailand. Macrotermes gilvus (Hagen) soldier termites were collected from 25 mounds. In four of the 25 mounds, scuttle fly larvae were found inside the bodies of the soldier termites, and adult flies were found in all of the mounds. Some of the larvae successfully developed to pupae under laboratory conditions. The percentages of parasitized major soldier termites collected from the four mounds were 43.79%, 47.43%, 0.86%, and 3.49%, respectively, and the percentages of parasitized minor soldier termites were 0.64%, 0.00%, 0.21%, and 0.00%, respectively. Larvae, pupae, and adult flies were identified using both morphological and molecular identifications. Molecular identification used the partial nucleotide sequences of the mitochondrial cytochrome c oxidase I (COI) gene. The results of both identification methods identified the parasitic Diptera as the scuttle fly, Megaselia scalaris (Loew) (Diptera: Phoridae). The phylogenetic analysis of the 23 scuttle fly samples (11 larvae, 7 pupae, and 5 adults) classified them into two clades: (1) Those closely related to a previous report in India; (2) those related to M. scalaris found in Asia and Africa. This is the first discovery of M. scalaris in M. gilvus. Further investgation into termite parasitism by M. scalaris and its possible use in the biological control of termites is needed.