Endophytic bacteria were isolated from Citrus plants and based on the similarity of 16S rRNA gene sequence, 20 isolates were included in the genus Curtobacterium in the family Microbacteriaceae, class Actinobacteria. Amplified Fragment Length Polymorphism (AFLP) analysis indicated that these strains formed four clusters with low variability that were separated from Curtobacterium flaccumfaciens. The isolates were Gram-positive, non-motile, non-spore forming, pale-yellow to orange-pink colonies. Analysis of eleven isolates showed that the major fatty acids of these strains were anteiso-C15:0, anteiso-C17:0 and iso-C16:0 and MK-9 and MK-8 as the major isoprenoid quinone, supporting the affiliation into Curtobacterium genus. The similarity of 16S rRNA gene between these isolates ranged from 99.9 to 100%, suggesting that this endophytic population present a low variability and similarity to species with validity published names within this genus, forming a distinct group in the phylogenetic tree. The DNA–DNA relatedness values to closest species were less than 48% for ER1/6T, suggesting that this strain does not belong to already described species. Analysis of the ER1/6T genome detected genes predicted to be involved in secondary metabolites synthesis, such as siderophore Desferrioxamine-like, bacteriocin Lactococcin 972-like, and terpene C50 carotenoid-like. Based on genome sequencing, the G + C content of the strain ER1/6T was 72.2%. Therefore, the polyphasic taxonomic characterization demonstrated that the strains ER1/6T, ER1.4/2, SR4/1 and SR4/8 dominant in the citrus tissues, represent a new species of the genus Curtobacterium, for which the name Curtobacterium uspiensis sp. nov. is proposed, with strain ER1/6T = CBMAI 2131 as the type strain.