In the present study, the urban wastewater sludges were treated in two-phase separate anaerobic digesters. The first digester was used for hydrolysis, acidification and acetogenesis, whereas the second one was for methanogenesis The two series of digesters were fed with the same raw sludge and working in steady-state conditions. Sludge samples were taken daily from both digesters for analyses. Special focus was placed on characterization of organic matter using diffuse reflectance infrared transform spectroscopy (DRIFT), 13 C stable isotopic ratio, differential thermal (DTA) analyses. The composition of volatile (VFA) was also analyzed, by head-space solid phase microextraction combined with gas-chromatography and mass spectrometry (HS-SPME-GC-MS), and discussed. Valuable information about the phase of degradation or stabilization of sludges was obtained by means of these analytical techniques, suggesting the possibility of a more efficient recycling of urban wastewaters organic matter.