Approximately 30 million years ago, ancestors of fungus-growing termites started an obligate mutualistic relationship with a Basidiomycete fungus Termitomyces. The success of this obligate relation is the division of labour and reliance on termite caste gut microbial symbionts. Termites workers maintain Termitomyces fungal garden with their workforce and dual gut passage while the soldier caste protects the colony from predators. The fungal garden concurrently provides enough food for the colony members. Royal pair (a king and a queen) is the centralised caste in the colony, and they control the colony population by their massive reproduction, but their gut community composition remains unexplored. This project aimed to characterise the gut microbes associated with royal pairs of a fungus‐growing termite species Macrotermes natalensis. Four colonies were explored using high throughput sequencing of 16S rRNA gene amplicon dataset. The high-throughput sequence result showed that royal gut microbiotas were comprised of a lower number of bacterial taxa than sterile caste (workers and soldiers). This less number of bacterial taxa suggested that the royal pair gut was completely decoupled from the sterile castes gut, which indicates that the royal pair were possibly provided with a unique diet. The study also showed diversity in bacterial genus-level OTUs of royal pairs in all four colonies which indicated that there is a diet variation between the king and queen. The media predicting strategy could facilitate future cultivation efforts for targeted royal pair gut bacterial strains.