The precious red coral Corallium rubrum (L., 1758) lives in the Mediterranean Sea and adjacent Eastern Atlantic Ocean on subtidal hard substrates. Corallium rubrum is a long-lived gorgonian coral that has been commercially harvested since ancient times for its red axial calcitic skeleton and which, at present, is thought to be in decline because of overexploitation. The depth distribution of C. rubrum is known to range from c. 15 to 300 m. Recently, live red coral colonies have been observed in the Strait of Sicily at depths of c. 600–800 m. This record sheds new light on the ecology, biology, biogeography and dispersal mechanism of this species and calls for an evaluation of the genetic divergence occurring among highly fragmented populations. A genetic characterization of the deep-sea red coral colonies has been done to investigate biological processes affecting dispersal and population resilience, as well as to define the level of isolation/differentiation between shallow- and deep-water populations of the Mediterranean Sea. Deep-water C. rubrum colonies were collected at two sites (south of Malta and off Linosa Island) during the cruise MARCOS of the R/V Urania. Collected colonies were genotyped using a set of molecular markers differing in their level of polymorphism. Microsatellites have been confirmed to be useful markers for individual genotyping of C. rubrum colonies. ITS-1 and mtMSH sequences of deep-water red coral colonies were found to be different from those found in shallow water colonies, suggesting the possible occurrence of genetic isolation among shallow- and deep-water populations. These findings suggest that genetic diversity of red coral over its actual range of depth distribution is shaped by complex interactions among geological, historical, biological and ecological processes.