Tropomyosins (Tms) are a family of highly conserved actin-binding proteins that play critical roles in a variety of processes, most notably, in the regulation of muscle contraction and relaxation. It is well known that different Tm isoforms have distinct functions and that altered expression of Tm isoforms could lead to changes in cardiac structure and function. To precisely define Tm isoform expression in the human heart, towards a better understanding of their functional roles, we have employed top-down mass spectrometry for in-depth proteomic characterization of Tm isoforms. Using a minimal amount of human heart tissue from rejected donor organs, we confirmed the presence of multiple Tm isoforms including α-Tm, β-Tm and κ-Tm in the human heart, with α-Tm being the predominant isoform, followed by minor isoforms of β-Tm and κ-Tm. Interestingly, our data revealed regional variations of Tm isoforms and post-translational modifications in the human heart. Specifically, the expression level of κ-Tm was highest in the left atrium but nearly undetectable in the left ventricle. The phosphorylation level of α-Tm (pα-Tm) was significantly higher in the atria than it was in the ventricles. The sequences of all Tm isoforms were characterized and the sites of post-translational modifications were localized. Clearly, top-down mass spectrometry is an attractive method for comprehensive characterization of Tm isoforms and post-translational modifications since it can universally detect and quantify all types of protein modifications without a priori knowledge and without the need for specific antibodies.