<p>Congenital Heart Disease (CHD) is currently the most common birth defect, affecting approximately 1% of live births. Understanding the genetic mechanisms underlying CHD is crucial for developing effective interventions. This paper explores the functional and evolutionary roles of the T-box transcription factors Tbx2a and Tbx2b in zebrafish heart development. Functional studies in zebrafish demonstrated that both homozygous and heterozygous mutations in <i>tbx2a</i> and <i>tbx2b</i> result in heart looping defects, challenging the assumption of their redundancy and indicating unique, non-overlapping functions essential for cardiac development. The observed phenotypic variability in heterozygous mutants suggests a complex interplay between these genes and highlights the sensitivity of cardiac development to precise gene dosage. Utilizing advanced bioinformatics techniques, we reconstructed the ancestral sequences of these genes to understand their evolutionary trajectory and functional divergence. Our analysis revealed strong evolutionary conservation of the T-box DNA-binding domain in Tbx2a and Tbx2b, suggesting that both proteins remain constrained to recognize the same core cis-regulatory elements. This conservation implies that their ability to bind essential cardiac target genes is functionally indispensable and under strong purifying selection. We observed unique and shared amino acid substitutions, indicating potential adaptive changes and conserved functions. Given their highly conserved DNA-binding domains, we hypothesized that Tbx2a and Tbx2b interact with different nuclear factors to regulate distinct sets of genes. Mass spectrometry-based proteomics provided insights into the unique nuclear interactions of Tbx2a and Tbx2b, supporting the hypothesis of their functional divergence. Overall, this research offers new insights into the functional and evolutionary roles of Tbx2a and Tbx2b in heart development, with implications for understanding the genetic basis of CHD.</p>