The meibomian and sebaceous glands secrete lipids to prevent desiccation of the ocular surface and skin, respectively. Precisely how these holocrine tissues regenerate is not well understood. To address this, we characterized keratin 5+ (K5) label-retaining cells (LRCs) and the lineage tracing of keratin 14 (K14) progenitors in mouse meibomian glands. Using the tet-off H2B-GFP/K5tTA mouse, H2B-GFP fluorescence dilutes 2-fold with every division in K5+ cell nuclei after doxycycline administration. In 3D reconstructions generated over a >28-day doxycycline chase, we observed LRCs at the acinus entrance where K6+ ductal epithelium terminates. For lineage tracing, K14CreERT2-Confetti mice were injected intraperitoneally with tamoxifen and euthanized at 23 and 59 weeks later. Meibomian gland acini in these mice were either monochromatic or dual-colored, whereas the duct exhibited multiple colors. In conclusion, LRCs are likely to direct meibomian gland turnover and may exist as two distinct unipotent progenitors that renew ductal and acinar tissue separately.