An essential step in the biosynthesis of polyketide and non-ribosomal peptide natural products is cleavage of the thioester bond that tethers the acyl/peptidyl chain to its biosynthetic enzyme. In modular polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) chain release is typically catalysed by a single C-terminal thioesterase domain. A clear exception is the bimodular PKS-NRPS BurA that produces gonyol-an intermediate in the biosynthesis of the cytotoxic Burkholderia virulence factor malleicyprol. While BurA lacks a C-terminal thioesterase domain, making the mechanism by which gonyol is released unclear, it contains two uncommon non-C-terminal thioesterase domains: one at the N-terminus of module one (BurA TE-A) and one within module two (BurA TE-B). Here we show using a sequence similarity network and site-directed mutagenesis that BurA TE-A resembles proofreading type II thioesterases and is not essential for gonyol biosynthesis, indicating a hydrolytic proofreading role. In contrast, the intramodular BurA TE-B is essential and catalyses the hydrolytic release of gonyol. Furthermore, unlike typical type I thioesterase domains, BurA TE-B accepts its acyl substrate from a downstream carrier-protein domain as opposed to an upstream one. Our findings clarify an important step in malleicyprol biosynthesis, reveal the flexibility of thioesterase domain positioning, and will serve as a basis for understanding other intramodular thioesterase domains.