In both unicellular and multicellular organisms, long-tailed class I myosins function in clathrin-mediated endocytosis. Myosin 1e (Myo1e) in vertebrates and Myo1 in fission yeast have similar domain organization, yet whether these proteins or their individual protein domains are functionally interchangeable remains unknown. In an effort to assess functional conservation of class I myosins, we tested whether human Myo1e could replace Myo1 in fission yeast Schizosaccharomyces pombe and found that it was unable to substitute for yeast Myo1. To determine if any individual protein domain is responsible for the inability of Myo1e to function in yeast, we created human-yeast myosin-I chimeras. By functionally testing these chimeric myosins in vivo, we concluded that the Myo1e motor domain is unable to function in yeast, even when combined with the yeast Myo1 tail and a full complement of yeast regulatory light chains. Conversely, the Myo1e tail, when attached to the yeast Myo1 motor domain, supports localization to endocytic actin patches and partially rescues the endocytosis defect in myo1Δ cells. Further dissection showed that both the TH1 and TH2-SH3 domains in the human Myo1e tail are required for localization and function of chimeric myosin-I at endocytic sites. Overall, this study provides insights into the role of individual myosin-I domains, expands the utility of fission yeast as a simple model system to study the effects of disease-associated MYO1E mutations, and supports a model of co-evolution between a myosin motor and its actin track.