ABSTRACT Mycobacterium abscessus is an emerging pathogen against which clarithromycin is the main drug used. Clinical failures are commonly observed and were first attributed to acquired mutations in rrl encoding 23S rRNA but were then attributed to the intrinsic production of the erm (41) 23S RNA methylase. Since strains of M. abscessus were recently distributed into subspecies and erm (41) sequevars, we investigated acquired clarithromycin resistance mechanisms in mutants selected in vitro from four representative strains. Mutants were sequenced for rrl , erm (41), whiB , rpIV , and rplD and studied for seven antibiotic MICs. For mutants obtained from strain M. abscessus subsp. abscessus erm (41) T28 sequevar and strain M. abscessus subsp. bolletii , which are both known to produce effective methylase, rrl was mutated in only 19% (4/21) and 32.5% (13/40) of mutants, respectively, at position 2058 (A2058C, A2058G) or position 2059 (A2059C, A2059G). No mutations were observed in any of the other genes studied, and resistance to other antibiotics (amikacin, cefoxitin, imipenem, tigecycline, linezolid, and ciprofloxacin) was mainly unchanged. For M. abscessus subsp. abscessus erm (41) C28 sequevar and M. abscessus subsp. massiliense , not producing effective methylase, 100% (26/26) and 97.5% (39/40) of mutants had rrl mutations at position 2058 (A2058C, A2058G, A2058T) or position 2059 (A2059C, A2059G). The remaining M. abscessus subsp. massiliense mutant showed an 18-bp repeat insertion in rpIV , encoding the L22 protein. Our results showed that acquisition of clarithromycin resistance is 100% mediated by structural 50S ribosomal subunit mutations for M. abscessus subsp. abscessus erm (41) C28 and M. abscessus subsp. massiliense , whereas it is less common for M. abscessus subsp. abscessus erm (41) T28 sequevar and M. abscessus subsp. bolletii , where other mechanisms may be responsible for failure.