Abstract: Neutrophils mediate an expanding range of homeostatic and pathologic functions. Correspondingly they exhibit extensive phenotypic heterogeneity, but bona fide neutrophil subsets remain controversial. Here we applied single-cell RNA sequencing to >17,000 healthy and perturbed murine neutrophils across biological tissues. Depending on modeling assumptions, neutrophils segregated into between 3 and 23 transcriptomic clusters. Using diffusion maps and RNA velocity analysis, we could instead model gene expression along a single chronologically-ordered developmental spectrum, termed here neutrotime. Neutrotime extends from immature Ly6ghigh Camphigh Ltfhigh Lcn2high neutrophils, found in greatest abundance in the bone marrow, to Il1bhigh Ccl6high Csf3rhigh neutrophils that predominate in blood and spleen. Data from developing neutrophils and ex vivo-differentiated neutrophils integrated smoothly into neutrotime, further validating the model. Experimental inflammation of lung, peritoneum and joint translated into de novo transcriptional activity reflecting structured permutations of the neutrotime paradigm. Our data establish a resource that captures the transcriptional diversity of neutrophils across tissues and inflammatory conditions on a single-cell level, defining a single maturational spectrum as the dominant organizational theme driving neutrophil heterogeneity. Authors: Ricardo Grieshaber-Bouyer (1, 2), Felix Radtke (1, 2), Pierre Cunin (1), Giuseppina Stifano (1), Anais Levescot (1), Brinda Vijaykumar (3), Nathan Nelson-Maney (1), Rachel B. Blaustein (1), Paul A. Monach (1, 4), Peter A. Nigrovic (1, 5), ImmGen Consortium