Heart failure (HF) after myocardial infarction (MI) is a leading cause of death in the western world with a critical need for new therapies. A previously developed injectable hydrogel derived from porcine myocardial matrix (PMM) has had successful results in both small and large animal MI models. In this study, we sought to evaluate the impact of tissue source on this biomaterial, specifically comparing porcine and human myocardium sources. We first developed an analogous hydrogel derived from human myocardial matrix (HMM). The biochemical and physical properties of the PMM and HMM hydrogels were then characterized, including residual dsDNA, protein content, sulfated glycosaminoglycan (sGAG) content, complex viscosity, storage and loss moduli, and nano-scale topography. Biochemical activity was investigated with in vitro studies for the proliferation of vascular cells and differentiation of human cardiomyocyte progenitor cells (hCMPCs). Next, in vivo gelation and material spread were confirmed for both PMM and HMM after intramyocardial injection. After extensive comparison, the matrices were found to be similar, yet did show some differences. Because of the rarity of collecting healthy human hearts, the increased difficulty in processing the human tissue, shifts in ECM composition due to aging, and significant patient-to-patient variability, these studies suggest that the HMM is not a viable option as a scalable product for the clinic; however, the HMM has potential as a tool for in vitro cell culture.