Tracking mature neutrophils remains challenging due to the lack of reliable cell surface markers. Although CD101 is a promising candidate as a marker for mature neutrophils, its stability under pathological conditions is unclear. Here, we developed a novel CD101-tdTomato reporter mouse model to specifically identify mature neutrophils. CD101 expression, tdTomato fluorescence, and neutrophil physiology were characterized by flow cytometry across peripheral tissues under homeostasis and by a transwell migration assay, in parallel with morphological features assessed by Wright-Giemsa staining. To assess marker stability, CD101-tdTomato and wild-type (WT) mice were treated with lipopolysaccharide (LPS) (200 ng, intraperitoneally, 2 h, n = 8), influenza virus (5 × 10³ PFU/mL, intratracheally, days 1 and 5, n = 6), or granulocyte colony-stimulating factor (G-CSF) (2.5 μg, intravenously, 2–6 h, n = 5), followed by flow cytometry analyses. CD101-tdTomato mice were crossed with Lysozyme-GFP mice to validate reporter specificity across myeloid subsets. We confirmed that CD101⁺ and tdTomato⁺ neutrophils display identical characteristics of mature neutrophils, including poly-segmented nuclei, cell size, and key functional properties under homeostasis. Under pathological challenges, the decrease in CD101 expression was not attributed to shedding or degradation of the protein, supporting its robustness under inflammatory conditions. Using CD101-tdTomato × Lysozyme-GFP mice, we established a dual-reporter system that enables precise distinction of mature neutrophils from total myeloid cells. Collectively, our findings support CD101 as a robust and reliable marker of neutrophil maturity, providing a foundation for its future applications in spatial transcriptomics and lineage tracing studies of the heterogeneity and functions of neutrophils.