Identifying the zinc (Zn) ligation and coordination environment in complex biological and environmental systems is crucial to understand the role of Zn as a biologically essential but sometimes toxic metal. Most studies on Zn coordination in biological or environmental samples rely on the extended X-ray absorption fine structure (EXAFS) region of a Zn K-edge X-ray absorption spectroscopy (XAS) spectrum. However, EXAFS analysis cannot identify unique nearest neighbors with similar atomic number (i.e., O versus N) and provides little information on Zn ligation. Herein, we demonstrate that high energy resolution-X-ray absorption near edge structure (HR-XANES) spectroscopy enables the direct determination of Zn ligation in whole cell bacteria, providing additional insights lost from EXAFS analysis at a fraction of the scan time and Zn concentration. HR-XANES is a relatively new technique that has improved our understanding of trace metals (e.g., Hg, Cu, and Ce) in dilute systems. This study is the first to show that HR-XANES can unambiguously detect Zn coordination to carboxyl, phosphoryl, imidazole, and/or thiol moieties in model microorganisms.