<p>Cardiomyocyte-specific Cre driver lines such as Myh6-Cre are widely used in cardiovascular research, but Cre recombinase itself can induce cardiac toxicity independently of target gene deletion. Early biomarkers capable of identifying this injury before overt cardiac remodeling or dysfunction remain poorly defined. Here, we performed a longitudinal analysis of Myh6-Cre mice using circulating biomarkers, cardiomyocyte Ca²⁺ transient measurements, histology, echocardiography, organ pathology, and survival analysis. Circulating cardiac troponin T (cTnT) levels were unchanged at 7 and 8 weeks of age but increased significantly from 9 weeks onward, preceding detectable structural remodeling or global cardiac dysfunction. At 12 weeks, isolated ventricular cardiomyocytes from Myh6-Cre mice showed reduced Ca²⁺ transient amplitude and delayed Ca²⁺ decay, indicating early functional impairment at the cellular level. Histologically detectable myocardial fibrosis emerged from 20 weeks, systolic dysfunction became evident at approximately 30 weeks, and progressive myocardial degeneration, extracardiac pathology, and mortality developed at later stages. These findings define the temporal progression of cardiac injury in Myh6-Cre mice and identify circulating cTnT as an early biomarker of cardiomyocyte injury caused by cardiac-specific Cre expression. Routine assessment of cTnT may improve the interpretation of phenotypes and help identify early model-associated injury in widely used Cre-based cardiac models, thereby informing the suitability of cardiac-specific Cre systems for cardiovascular research.</p>