We describe a sequence-guided approach to identify and engineer key amino acids in orthologous CYP4B1 enzymes for selective ω-hydroxylation of saturated fatty acids and sequential hydroxylation of trans-stilbene to trans-4,4′- and trans-4,3′-dihydroxystilbene. Orthologous enzymes generally share high sequence identities and catalyze the same reactions, but often exhibit subtle yet distinct activity and selectivity profiles. Understanding the sequence determinants that underlie this observation is not only essential for elucidation of evolutionary adaptations, but can also be used for rational biocatalyst design. Within this study, sequence comparison of a set of orthologous CYP4B1 enzymes identified four amino acids as key activity and selectivity drivers for catalysis. These were systematically engineered by introducing potentially beneficial single amino acid exchanges into less active/selective CYP4B1 orthologs, and vice versa. The exchanges shifted activity and selectivity trends for ω-hydroxylation of lauric acid by the mutated orthologs in a manner consistent with those observed for the corresponding wildtype enzymes across multiple orthologous backgrounds. Notably, combinatorial mutagenesis of two amino acids resulted not only in additive, but also in orthologous- and substrate-dependent adverse influences on activity and selectivity, which likely reflects the enzyme function optimized by nature in the context of the individual orthologs evolutionary background. For conversion of trans-stilbene, two beneficial amino acid exchanges between two CYP4B1 orthologs resulted in 95% conversion of trans-stilbene and 85% dihydroxystilbenes. In summary, we demonstrate that systematic introduction of only one or two sequence-guided mutations can be sufficient to substantially alter activity and selectivity profiles of orthologous enzymes for a given substrate, which highlights the usefulness of this approach for rational design of biocatalysts.