Sebum lipids, accessible via groomed latent fingerprints, may be a valuable, underappreciated sample source for future biomarker research. Sampling sebum lipids from the skin is painless for patients, efficient for researchers, and has already demonstrated the potential to contain disease biomarkers. However, before sebum sampling can be implemented in routine studies, more information is needed regarding sampling reproducibility and variability. This information will enable researchers to choose the best practices for sebum-based studies. Herein, we use our recently established workflow for the collection and analysis of groomed fingerprints to assess the reproducibility of lipid profiles obtained via mass spectrometry. Using 180 fingerprint samples collected from 30 participants, we also assess lipid changes according to biological sex and anatomical grooming region (cheek, neck, and forehead) via supervised and unsupervised classification. The results demonstrate that this sampling protocol achieves satisfactory reproducibility, and negligible differences exist between male and female groomed fingerprint lipids. Moreover, the anatomical grooming region can impact the fingerprint lipid profile: cheek- and forehead-groomed fingerprints are more similar to one another than either collection site is to neck-groomed fingerprints. This information will inform future sebum-based biomarker investigations, enabling researchers to collect meaningful lipidomic datasets from groomed fingerprint samples.