Testudine intranuclear coccidiosis (TINC) significantly impairs global chelonian conservation efforts and trade. The disease may present a clinical and diagnostic challenge due to variable, nonspecific clinical signs and cryptic to patchy distribution within lesions microscopically. The objective of this work was to develop a species-specific, in situ hybridization (ISH) assay for labelling organisms in formalin preserved, histologic tissues. Thirteen previously diagnosed TINC cases and ten histologically negative cases from three diagnostic laboratory archives involving eight chelonian species were selected for inclusion in this study. Tissues examined by ISH include stomach, intestine, liver, spleen, kidney, pancreas, larynx, trachea, esophagus, heart, and ovary. The probe targeted a variable region of the 18S rRNA sequence specific for TINC. The assay demonstrated well defined, specific labelling of organisms across multiple tissue types without cross reaction with host tissues or other pathogens in the sections. The development of this assay provides a powerful new tool for diagnostic investigation and answering future research questions about this impactful, enigmatic pathogen.