AbstractRifampicin resistance (Rif^R) is caused by mutations in rpoB, encoding the β-subunit of RNA polymerase. Rif^R mutations generally incur a fitness cost and in resistant isolates are frequently accompanied by compensatory mutations in rpoA, rpoB or rpoC. Previous studies of fitness compensation focused on Rif^R caused by amino acid substitutions within rpoB. Rif^R is also caused by deletion and duplication mutations in rpoB but it is not known whether or how such mutants can ameliorate their fitness costs. Using experimental evolution of Salmonella carrying Rif^R deletion or duplication mutations we identified compensatory amino acid substitution mutations within rpoA, rpoB or rpoC in 16 of 21 evolved lineages. Additionally, we found one lineage where a large deletion was compensated by duplication of adjacent amino acids (possibly to fill the gap within the protein structure), two lineages where mutations occurred outside of rpoABC, and two lineages where a duplication mutant reverted to the wild-type sequence. All but the two revertant mutants maintained the Rif^R phenotype. These data suggest that amino acid substitution mutations are the major compensatory mechanism regardless of the nature of the primary Rif^R mutation.