An actinomycete, strain D1T, was isolated from a freshwater sediment sample collected from the San Pablo river in the La Risueña community, Santiago de Cuba province, Cuba. The strain was identified as a member of the genus Nocardiopsis by means of a polyphasic taxonomic study. It produced a light yellow non-fragmented substrate mycelium, a white well-developed aerial mycelium and straight to flexuous hyphae. No specific spore chains were observed. Strain D1T contained meso-diaminopimelic acid, no diagnostic sugars, and MK-10(H2), MK-10(H4), MK-10 and MK-10(H6) as predominant menaquinones, but not phosphatidylcholine as diagnostic polar lipid of the genus Nocardiopsis. The predominant fatty acids were iso-C16 : 0, 10-methyl-C18 : 0 and anteiso-C17 : 0. Strain D1T showed the highest degree of 16S rRNA gene sequence similarity to Nocardiopsis synnematoformans DSM 44143T (99.8 %), Nocardiopsis dassonvillei subsp. albirubida NBRC 13392T (99.8 %) and Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111T (99.6 %). A genomic OrthoANIu value between D1T and N. dassonvillei subsp. dassonvillei DSM 43111T of 97.63 % and a dDDH value of 78.9 % indicated that strain D1T should be classified in N. dassonvillei. However, phenotypic characteristics distinguished strain D1T from its nearest neighbour taxon. On basis of these results we propose to classify strain D1T (=LMG 30468T=CECT 30033T) as a representative of a novel subspecies of the genus Nocardiopsis, for which the name Nocardiopsis dassonvillei subsp. crassaminis subsp. nov. is proposed. In addition, the genomic distance between N. dassonvillei subsp. albirubida NBRC 13392T and N. dassonvillei subsp. dassonvillei DSM 43111T as determined through OrthoANIu (93.64 %) and dDDH (53.40 %), along with considerable phenotypic and chemotaxonomic differences reported in earlier studies, indicated that the classification of this taxon as Nocardiopsis alborubida Grund and Kroppenstedt 1990 is to be preferred over its classification as N. dassonvillei subsp. albirubida Evtushenko et al. 2000.