Cardiac regeneration studies have been plagued by technical challenges in unequivocally identifying cardiomyocyte (CM) nuclei in cardiac sections, crucial for accurate identification of cycling CMs. The use of antibodies to sarcomeric proteins is error-prone, the CM specificity of common nuclear markers is controversial, and utilizing genetically modified mouse models poses risk of inducing unintended cardiac phenotypes. The application of RNAscope intronic probes overcomes the above shortcomings. Intronic probes label intronic RNAs within nuclei and can therefore be utilized as a method for nuclear localization. A Tnnt2 intronic RNAscope probe highly colocalized with Obscurin-H2B-GFP in adult mouse hearts, demonstrating CM specificity. Studies in embryos demonstrated that the Tnnt2 intronic RNAscope probe labeled CM nuclei that had undergone DNA replication, and remained closely associated with CM chromatin at all stages of mitosis, even with nuclear envelope breakdown. The efficiency, accuracy, and perdurance of the Tnnt2 intronic RNAscope probe even with nuclear envelope breakdown facilitated reliable investigation of dynamics of DNA synthesis and potential mitoses in CMs in both border and infarct zones after myocardial infarction (MI). Furthermore, we designed Myl2 and Myl4 intronic RNAscope probes, which labeled ventricular and atrial CM nuclei, respectively, and may help identify CM subtypes generated in vitro.