TEM-168 is a TEM-β-lactamase variant previously derived only in the laboratory. This study first identified this variant in a clinically isolated Escherichia coli strain N08-1503. Antimicrobial susceptibility testing was performed using disk diffusion, broth microdilution (per CLSI guidelines), and Etests. The strain was resistant to ampicillin, cefazolin, and ampicillin-sulbactam, with reduced susceptibility to ceftazidime; it tested negative for extended-spectrum β-lactamase (ESBL) by standard CLSI methods but positive for cefepime ESBL using Etest. PCR and sequence analysis revealed a blaTEM-168 gene encoding TEM-168, a TEM-1 variant with a T261M substitution (T265M by Ambler numbering) caused by a C782T transition. The blaTEM-168 promoter region had a G162T change defining the strong P4 promoter. A ~13-kb plasmid pT168 harboring blaTEM-168 was transformable into E. coli DH10B, and transformants exhibited similar resistance phenotypes (resistant to amoxicillin-clavulanic acid and piperacillin-tazobactam, intermediate to cefoxitin). Isoelectric focusing identified a single β-lactamase band with a pI of 5.4. Resistance to β-lactam-inhibitor combinations likely resulted from hyperproduction of TEM-168 via the P4 promoter. TEM-168 had not been previously reported in clinical isolates due to negative standard ESBL tests. The sequence of blaTEM-168 was assigned GenBank accession number FJ919776.