Introduction: Exosomes are extracellular vesicles 80-150 nm in diameter, containing proteins, mRNAs, microRNAs, and lipids reflecting the parent cell. While there has been an extensive characterization of the cargo incorporated in exosomes, a detailed morphological analysis of exosomes purified by various isolation techniques has not been performed. Objective: We aimed to determine the heterogeneity of exosomes morphology and if such morphological features are conserved across sample types. Methods: Our study used Cryogenic Electron Microscopy (Cryo-EM) to examine exosome size and morphology. Results: Our results revealed significant diversity in extracellular vesicle morphology independent of the isolation method, suggesting that morphological subpopulations of these vesicles exist. Based on their shape, our analysis classified exosomes into seven categories. In addition, we developed a semi-automatic image analysis framework to accurately characterize exosome attributes and distribution to facilitate reliable quantification of specific bio-nanoparticle features in Cryo-EM micrographs. Conclusions: Morphological features of exosomes inform their biophysical properties, which influence both biodistribution and biological activity in vivo. Our data demonstrating the innate morphological diversity of exosomes may have implications for improving the specificity and precision of exosome-delivered therapeutics. Conflict of interest: R.K. and MD Anderson Cancer Center hold patents in exosome biology and are stock equity holders in Codiak Biosciences Inc. R.K. is a consultant and a scientific advisor of Codiak Biosciences Inc.