Antibiotic tolerance is associated with antibiotic treatment failure, and molecular mechanisms underlying tolerance are poorly understood. We recently succeeded in inducing tolerance to ceftriaxone (CRO) in an N. gonorrhoeae reference isolate. In a prior in vitro study, six biological replicates of WHO P strains were exposed to CRO (10× the MIC) followed by overnight growth, and tolerance was assessed using a modified Tolerance Disc (T.D.) test. In the current study, we characterized the mutation profile of these CRO-tolerant phenotypes. The whole genome was sequenced from isolates from different replicates and time points. We identified mutations in four genes that may contribute to ceftriaxone tolerance in N. gonorrhoeae, including a mutation in the enolase (eno) gene that arose independently in three lineages.