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Endocytoscopy (EC) is an ultra-high magnification endoscopy designed to provide in vivo histologic assessment. This study aimed to evaluate the diagnostic yield of the newly developed fourth-generation EC for esophageal squamous lesions by using a modified EC classification. A total of 2548 EC images of 57 esophageal targeted areas between June 2015 and October 2017 were retrospectively collected. Two lesions with low-quality images were excluded. Only EC images were independently reviewed by two expert and two non-expert endoscopists.