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Cardiac troponin I (cTnI), a cardiac-specific biomarker used to detect myocardial injury in mammals, has only undergone cursory investigation in reptiles. This study’s objectives were to determine feasibility of cTnI detection in reptilian cardiac tissue, skeletal muscle, and plasma utilizing two different immunoassays, and compare tissue concentrations between immunoassays and taxonomic groups (chelonians, lizards, snakes). Tissue homogenates were created and total protein concentrations determined from cardiac tissue and skeletal muscle collected from 30 individual reptiles representing 25 different species. Heparinized plasma was collected from seven reptiles. Samples were analyzed on both point-of-care (i-STAT) and high-sensitivity (ADVIA) immunoassays when feasible, and standardized concentrations compared between sample type, immunoassay, and taxonomic group. cTnI was detected above the lower limit of detection (LoD) in cardiac tissues from 24/25 species on the i-STAT and 22/22 species on the ADVIA. Only one sample produced analyzer error, occurring on the i-STAT. Cardiac tissue had significantly higher cTnI in chelonians ( p adj = 0.034) and lizards ( p adj = 0.049) than snakes on the i-STAT, and in chelonians than both lizards ( p adj = 0.015) and snakes ( p adj < 0.01) on the ADVIA. The i-STAT and ADVIA had poor agreement for cardiac tissue samples. Most skeletal muscle samples reported analyzer error (20/30) or concentrations below the LoD (9/30) on the i-STAT. On the ADVIA all skeletal muscle samples were above the LoD (25/25), and four snakes had higher cTnI concentrations in skeletal muscle than cardiac tissue. All plasma samples had detectable cTnI on both immunoassays, but most on the i-STAT were below the LoD. At least 22 reptile species have detectable cTnI in cardiac tissue on two different immunoassays. Due to variability between taxonomic groups and immunoassays, establishment of both species-specific and assay-specific reference intervals are warranted for clinical utility.