Objectives: Mildred Trotter was an anatomist and physical anthropologist whose studies on hair morphology, growth, somatic distribution, and trait relationships to age and ethnogeographic population were foundational to the field of microscopical hair analysis. The collection of human hair samples she assembled for her research has been an underutilized resource for studies on human hair variation. We applied updated methods and reviewed Trotter's original data to reassess the relationship hair traits have to diverse population labels. Methods: Hair form and pigmentation patterns were measured from a subset of the hair samples accumulated by Trotter and we compared our data to Trotter's original results. Variability in hair traits were tested within individuals, within populations, and among ethnogeographic groups. Results: Measured hair cross-section dimensions and melanosome density and distribution revealed substantial variability within individuals and ethnogeographic populations. Hair traits were found to not be distinctly separable by ancestry but instead showed continuous variation across human populations. Trotter's measurements were precise and the dataset she compiled remains valid, though the conclusions should be reviewed in light of our current understanding of human variation. Discussion: Our findings support moving away from categorical ancestry classifications and eliminating the use of outdated racial typologies in favor of more descriptive trait analysis. Detailed analysis of trait pattern distributions are presented that may be useful for future research on human variation. We point to the need for additional research on human variation and hair trait relationships with reference to known population affinity.