We investigated extending the use of direct partial hsp65 gene sequencing for the identification of mycobacteria to isolates in primary liquid detection media as an economical, feasible, and more rapid means of identification. During the course of the study, the hsp65 sequence-based identifications for isolates from 670 primary liquid detection media determined to be positive for acid-fast bacilli were compared to the identifications derived from Accuprobes, biochemical test panels, or 16S rRNA gene sequencing. Preliminary analysis indicated a 97.6% concordance, with a final agreement of 99.1% between the identification algorithms. hsp65 sequencing costs (32.84 US dollars) were greater than the cost of identification with Accuprobe (9 US dollars) but less than the cost of the biochemical test panel identification (average cost, 98.90 US dollars) and equivalent to the cost of 16S rRNA sequencing, although there was a referral cost (59.85 US dollars) for the shipping of isolates to another reference laboratory. Analysis indicated that our laboratory would have recognized a cost savings of approximately 12,000 US dollars by using hsp65 sequencing to identify isolates from specimens with a negative fluorescent- smear status and would have achieved further savings by using it as an alternative to biochemical panel testing for fluorescent-smear-positive specimens. The time to identification by hsp65 gene sequencing was slightly longer than that required by the Accuprobe assay (1 versus 2 days), shorter than that required by the biochemical test panels (2 days versus 26 days on average), and more rapid than referral for 16S rRNA gene sequencing.