The subterranean termite Globitermes sulphureus (Blattodea: Termitidae) can be found in tropical regions. We chose seven novel species-specific microsatellite markers to infer the breeding pattern of G. sulphureus based on its colony and population genetic structure in eight selected populations (natural-n = 4 and metropolitan-n = 4) in Kedah and Penang, Malaysia. A strong correlation with their geographical location is shown by the acquired genetic gap for all studied populations from this study. The breeding pattern of family structure and comparisons of estimated F-statistics among G. sulphureus workers suggests 60% of all colonies are mixed families, whereas the remaining are simple families. Average relatedness values within simple and mixed family colonies are similar (r = 0.121). Positive fixation index FST values (FST = 0.086) indicate all eight populations (>500 m apart) have a significantly moderate genetic differentiation and low levels of inbreeding based on the low overall inbreeding coefficient FIT value of 0.391. Furthermore, four populations; Palapes USM (PU), Tmn Astana (TA), Kg Teluk (KT), and Penang National Park (NP), deviate from Hardy–Weinberg equilibrium (HWE, all p = 0.000) and five studied polymorphic loci (GS1, GS10, GS15, GS27 and GS29) are possibly under selection. The findings also reveal signs of a bottleneck effect in two populations: Tikam Batu (TB) and Penang National Park (NP), indicating genetic drift.
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