A Gram-stain-negative strain, A60T, isolated from a water well sample in Portugal, was characterized phenotypically, genotypically and phylogenetically. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain A60T belonged to the genus Citrobacter, and recN gene phylogeny revealed one strongly supported clade encompassing strain A60T and 13 other strains from public databases, distinct from currently recognized species of the genus Citrobacter. Furthermore, multilocus sequence analysis (MLSA) based on concatenated partial fusA, leuS, pyrG and rpoB sequences confirmed the classification obtained with the recN sequence. In silico genomic comparisons, including average nucleotide identity (ANI) and the genome-to-genome distance calculator (GGDC), showed 94.6 % and 58.4 % identity to the closest relative Citrobacter freundii ATCC 8090T, respectively. The ability to metabolize different compounds further discriminated strain A60T from other species of the genus Citrobacter. The G+C content of strain A60T is 52.0 %. The results obtained support the description of a novel species within the genus Citrobacter, for which the name Citrobacter portucalensis sp. nov. is proposed, with the type strain A60T (=DSM 104542T=CECT 9236T).