Mezcal is the second most important alcoholic beverage produced in Mexico, and the microorganisms present during the fermentation process are primarily responsible for its organoleptic characteristics. Among these, the bacterial populations have been less studied, mainly due to the difficulty of recovering them. The aim was to apply a culture-independent molecular technique to detect the bacterial populations involved in spontaneous mezcal fermentation in Durango State. Samples were obtained from several stages in the mezcal production line. Denaturing gradient gel electrophoresis (DGGE) analysis was performed using DNA extracted from isolated microorganisms and metagenomes obtained from samples directly taken from the fermentation process. This study indicated that non-isolated bacteria were significant in the fermentation process and indicated that the lactic acid and acetic acid bacterial populations present in the monitored process are similar to those present in other regions during fermentation, although they differ in some species that may play essential roles in the development of the typical organoleptic properties of mezcal produced in the State of Durango. Furthermore, this study indicates that culture-independent analysis by DGGE can reveal the bacterial diversity during the mezcal fermentation process without the use of complex isolation procedures and can be a useful tool for the analysis, monitoring and control of mezcal fermentation processes.