Two groups of 5 lambs were euthanized at the weaning (T45) and fattening stages (T90) to evaluate the use of microbial ribosomal DNA (rDNA) sequences as potential microbial markers in relation to purine bases (PB) as a conventional marker. Both microbial markers originated similar microbial N concentrations (mg/g of DM), although T45 showed decreased values compared with the T90 group when either PB or rDNA were considered (P = 0.02). The survival of microbial rDNA was determined in 3 digestive sites (omasum, abomasum, and duodenum), but no substantial differences were observed, indicating that rDNA maintains the molecular stability along the sampling sites analyzed. Contrarily PB concentration increased successively along the digestive tract (P < 0.05), likely as a consequence of the endogenous PB secretion. Undegraded milk PB may also explain the overestimation of the microbial N concentration (2.8 times greater) using PB than rDNA sequences. Abomasum was the sampling site where the best agreement between PB and rDNA estimations was observed. Protozoal N concentration was irrelevant in T45 animals, although substantial in T90 lambs (18% of microbial N). In conclusion, bacterial 16S and protozoal 18S rDNA sequences may persist through the gastric digestive tract and their utilization as a highly specific microbial marker should not be neglected.