The 3‐(3‐hydroxyalkanoyloxy)alkanoate (HAA) synthase RhlA is an essential enzyme involved in the biosynthesis of HAAs in Pseudomonas and Burkholderia species. RhlA modulates the aliphatic chain length in rhamnolipids, conferring distinct physicochemical properties to these biosurfactants exhibiting promising industrial and pharmaceutical value. A detailed molecular understanding of substrate specificity and catalytic performance in RhlA could offer protein engineering tools to develop designer variants involved in the synthesis of novel rhamnolipid mixtures for tailored eco‐friendly products. However, current directed evolution progress remains limited due to the absence of high‐throughput screening methodologies and lack of an experimentally resolved RhlA structure. In the present work, we used comparative modeling and chimeric‐based approaches to perform a comprehensive semi‐rational mutagenesis of RhlA from Pseudomonas aeruginosa. Our extensive RhlA mutational variants and chimeric hybrids between the Pseudomonas and Burkholderia homologs illustrate selective modulation of rhamnolipid alkyl chain length in both Pseudomonas aeruginosa and Burkholderia glumae. Our results also demonstrate the implication of a putative cap‐domain motif that covers the catalytic site of the enzyme and provides substrate specificity to RhlA. This semi‐rational mutant‐based survey reveals promising ‘hot‐spots’ for the modulation of RL congener patterns and potential control of enzyme activity, in addition to uncovering residue positions that modulate substrate selectivity between the Pseudomonas and Burkholderia functional homologs.DatabaseModel data are available in the PMDB database under the accession number PM0081867.