Aims: Investigation of the alkane-degrading properties of Dietzia sp. HOB, one of the isolated Corynebacterineae strains that became dominant after the Prestige oil spill. Methods and Results: Using molecular and chemical analyses, the alkane-degrading properties of strain Dietzia sp. HOB were analysed. This Gram-positive isolate was able to grow on n-alkanes ranging from C(12) to C(38) and branched alkanes (pristane and phytane). 8-Hexadecene was detected as an intermediate of hexadecane degradation by Dietzia HOB, suggesting a novel alkane-degrading pathway in this strain. Three putative alkane hydroxylase genes (one alkB homologue and two CYP153 gene homologues of cytochrome P450 family) were PCR-amplified from Dietzia HOB and differed from previously known hydroxylase genes, which might be related to the novel degrading activity observed on Dietzia HOB. The alkane degradation activity and the alkB and CYP153 gene expression were observed constitutively regardless of the presence of the substrate, suggesting additional, novel pathways for alkane degradation. Conclusions: The results from this study suggest novel alkane-degrading pathways in Dietzia HOB and a genetic background coding for two different putative oil-degrading enzymes, which is mostly unexplored and worth to be subject of further functional analysis. Significance and Impact of the Study: This study increases the scarce information available about the genetic background of alkane degradation in genus Dietzia and suggests new pathways and novel expression mechanisms of alkane degradation.