The variability of eosinophilic infiltrates in eosinophilic esophagitis is not well described. This study aimed to determine the distribution of esophageal eosinophilia and the utility of histologic cut-points for eosinophilic esophagitis diagnosis in subjects undergoing endoscopy. We performed a prospective study of adults undergoing outpatient endoscopy. Research protocol esophageal biopsies were obtained from all subjects. Incident cases of eosinophilic esophagitis were diagnosed per consensus guidelines. Biopsies were interpreted following a validated protocol, and maximum eosinophil counts (eosinophils per high-power field; eos/hpf) were determined. Histologic analyses were performed on a per-patient, per-biopsy, and per-hpf basis. There were 213 patients, yielding 923 esophageal biopsies with 4588 hpfs. Overall, 48 patients (23%), 165 biopsy fragments (18%), and 449 hpfs (10%) had ≥15 eos/hpf; most subjects had no or low levels of eosinophils. In the eosinophilic esophagitis cases, 119 biopsy fragments (63%) and 332 hpfs (36%) had ≥15 eos/hpf. There was a mean 104-fold difference between the lowest and highest hpf eosinophil count for the eosinophilic esophagitis patients; 85% of the biopsies from eosinophilic esophagitis cases also had at least one hpf with <15 eos/hpf. The cut-point of 15 eos/hpf had a sensitivity of 100% and a specificity of 96% for diagnosis of eosinophilic esophagitis. In conclusion, most patients have little to no esophageal eosinophilia. In patients with eosinophilic esophagitis, there was marked variability in the eosinophil counts by biopsy and by hpf within a given biopsy. Additionally, the 15 eos/hpf cut-point was highly sensitive and specific for eosinophilic esophagitis. Multiple esophageal biopsies from different locations should be obtained to optimize eosinophilic esophagitis diagnosis.