We compared and analyzed 16S rRNA and tuf gene sequences for 97 clinical isolates of coagulase-negative staphylococci (CNS) by use of the GenBank, MicroSeq, EzTaxon, and BIBI databases. Discordant results for definitive identification were observed and differed according to the different databases and target genes. Although higher percentages of sequence identity were obtained with GenBank and MicroSeq for 16S rRNA analysis, the BIBI and EzTaxon databases produced less ambiguous results. Greater discriminatory power and fewer multiple probable identifications were observed with tuf gene analysis than with 16S rRNA analysis. The most pertinent results for tuf gene analysis were obtained with the GenBank database when the cutoff values for the percentage of identity were adjusted to be greater than or equal to 98.0%, with >0.8% separation between species. Analysis of the tuf gene proved to be more discriminative for certain CNS species; further, this method exhibited better distinction in the identification of CNS clinical isolates.