The bacterial population during malolactic fermentation of Tempranillo wine was studied using the polymerase chain reaction-denaturing gradient gel electrophoresis, a culture-independent method successfully used for identification and monitoring of bacterial population in different habitats included food fermentations. The results showed that Oenococcus oeni was the predominant species in the malolactic fermentation of Tempranillo wines, although the presence of Gluconobacter oxydans, Asaia siamensis, Serratia sp., and Enterobacter sp. was also observed. These results were partly coincidental with those obtained from a culture-dependent method, using a selective medium. Therefore, it may be concluded that for a more complete knowledge of the bacterial community present during malolactic fermentation of Tempranillo wine, an approach that combines a culture-independent method and a culture-dependent method would be advisable.