Fermenters are widely used to study ruminal fermentation, but information on microbial populations developing in fermenters over the incubation period is limited. Four Rusitec fermenters were fed 2 diets representative of those administered to dairy sheep (DAI; 50:50 alfalfa hay:concentrate) and fattening lambs (FAT; 15:85 barley straw:concentrate) in a crossover design with 2 14-d incubation periods to assess the evolution of the microbial populations. There were 4 fermenters per diet. The fermenters received daily 30 g of diet DM and samples from liquid (LIQ) and solid (SOL) digesta were taken on d 3, 8 and 14, and stored frozen at −80ªC until DNA extraction. Concentrations of bacterial and protozoal DNA and relative abundance of fungi and methanogenic archaea to total bacterial DNA concentration were determined by real time PCR using previously validated primers and DNA from bacteria and protozoa isolated from sheep rumen as standards. Data were analyzed as a mixed model with repeated measures using the PROC MIXED of SAS. The model included diet, incubation run, time, and diet × time as fixed effects, and fermenter as a random effect. Diet x sampling time interactions (P > 0.05) were detected for bacterial and protozoal DNA concentrations in both digesta phases. The bacterial DNA concentrations in SOL did not change (P = 0.002) over the incubation period, whereas concentrations in LIQ increased (P < 0.001) by 1.5 and 1.8 times for DAI and FAT diets by the end of the incubation, respectively. Protozoal DNA concentrations on d 14 were 37.8 and 8.0 times lower (P < 0.001; means across diets) than those on d 3 for SOL and LIQ phases, respectively. Relative abundance of fungi decreased (P < 0.05) with time in both phases, and that of methanogenic archaea remain unchanged in LIQ and increased (P = 0.021) in SOL. Concentration of bacterial and protozoal DNA and the relative abundance of methanogenic archaea were greater in the fermenters fed the DAI diet (P < 0.05) compared with FAT diet. The results show that microbial populations in Rusitec fermenters are affected by the incubated diet and change over the incubation period.