Abstract <p>An important task on the way to creating a competent cellular equivalent of a biological heart pacemaker for the treatment of cardiovascular diseases is to assess the ability of cultured cardiomyocytes in conglomerates, layers, or pseudo-tissues to automatic, spontaneous activity. It is known that the electrophysiological and tissue properties of cultured cardiomyocyte structures differ significantly from native ones. In this work, based on a comparison of the transcription profile and an assessment of the expression level of key genes in the tissues of the sinoatrial node (SAN), atrium, neonatal cardiomyocytes, and cultured cardiomyocyte conglomerates of rats, pacemaker competence indices (PCI) are proposed, characterizing the ability (competence) of cellular structures to function as a pacemaker. Based on the analysis of mRNA sequencing data, it was found that there is a significant change in the expression profile of functionally significant genes during neonatal cardiomyocytes’ cultivation, which enhances the difference in the phenotype of emerging cardiomyocyte conglomerates from both the pacemaker of SAN and the contractile myocardium. It has been shown for the first time that satisfactory PCIs can be obtained based on an assessment of the transcript level of no more than four genes, including pairs of transcription factors (<i>Tbx3</i>, <i>Tbx18</i> and <i>Nkx2.5</i>, <i>Gata4</i>) or pairs of ion channels (<i>Hcn4</i>, <i>Kcnj2</i>) and gap junctions (<i>Gjc1</i>, <i>Gja1</i>) antagonistically expressed in the SAN and contractile myocardium. The PCI simplifies and accelerates the assessment of the functional properties of artificial spontaneously active cardiomyocyte cell structures.</p>