High-resolution melting (HRM) analysis followed by sequencing was applied for determination of bacteria grown on plates isolated from farmed mussels (Mytilus galloprovincialis) during their storage at 4 °C. The V3-V4 region of the 16S rRNA gene from the isolates was amplified using 16S universal primers. Melting curves (peaks) and high resolution melting curves (shape) of the amplicons and sequencing analysis were used for differentiation and identification of the isolated bacteria, respectively. The majority of the isolates (a sum of 101 colonies, from five time intervals: day 0, 2, 4, 6 and 8) from non-selective solid medium plates were classified in four bacterial groups based on the melting curves (peaks) and HRM curves (shape) of the amplicons, while three isolates presented distinct HRM curve profiles (single). Afterwards, sequencing analysis showed that the isolates with a) the same melting peak temperature and b) HRM curves that were >95% similar grouped into the same bacterial species. Therefore, based on this methodology, the cultivable microbial population of chill-stored mussels was initially dominated by Psychrobacter alimentarius against others, such as Psychrobacter pulmonis, Psychrobacter celer and Klebsiella pneumoniae. P. alimentarius was also the dominant microorganism at the time of the sensory rejection (day 8). Concluding, HRM analysis could be used as a useful tool for the rapid differentiation of the bacteria isolated from mussels during storage, at species level, and then identification is feasible by the sequencing of one only representative of each bacterial species, thus reducing the cost of required sequencing.