Four mesophilic, neutrophilic, and aerobic marine ammonia-oxidizing archaea, designated strains SCM1T, HCA1T, HCE1T and PS0T, were isolated from a tropical marine fish tank, dimly lit deep coastal waters, the lower euphotic zone of coastal waters, and near-surface sediment in the Puget Sound estuary, respectively. Cells are straight or slightly curved small rods, 0.15–0.26 µm in diameter and 0.50–1.59 µm in length. Motility was not observed, although strain PS0T possesses genes associated with archaeal flagella and chemotaxis, suggesting it may be motile under some conditions. Cell membranes consist of glycerol dibiphytanyl glycerol tetraether (GDGT) lipids, with crenarchaeol as the major component. Strain SCM1T displays a single surface layer (S-layer) with p6 symmetry, distinct from the p3-S-layer reported for the soil ammonia-oxidizing archaeon Nitrososphaera viennensis EN76T. Respiratory quinones consist of fully saturated and monounsaturated menaquinones with 6 isoprenoid units in the side chain. Cells obtain energy from ammonia oxidation and use carbon dioxide as carbon source; addition of an α-keto acid (α-ketoglutaric acid) was necessary to sustain growth of strains HCA1T, HCE1T, and PS0T. Strain PS0T uses urea as a source of ammonia for energy production and growth. All strains synthesize vitamin B1 (thiamine), B2 (riboflavin), B6 (pyridoxine), and B12 (cobalamin). Optimal growth occurs between 25 and 32 °C, between pH 6.8 and 7.3, and between 25 and 37 ‰ salinity. All strains have a low mol% G+C content of 33.0–34.2. Strains are related by 98 % or greater 16S rRNA gene sequence identity, sharing ~85 % 16S rRNA gene sequence identity with Nitrososphaera viennensis EN76T. All four isolates are well separated by phenotypic and genotypic characteristics and are here assigned to distinct species within the genus Nitrosopumilus gen. nov. Isolates SCM1T (=ATCC TSD-97T =NCIMB 15022T), HCA1T (=ATCC TSD-96T), HCE1T (=ATCC TSD-98T), and PS0T (=ATCC TSD-99T) are type strains of the species Nitrosopumilus maritimus sp. nov., Nitrosopumilus cobalaminigenes sp. nov., Nitrosopumilus oxyclinae sp. nov., and Nitrosopumilus ureiphilus sp. nov., respectively. In addition, we propose the family Nitrosopumilaceae fam. nov. and the order Nitrosopumilales ord. nov. within the class Nitrososphaeria .