Pre-treatment of biodegradable waste significantly affects the process of anaerobic digestion. This pre-treatment of biological materials lies in improving of the accessibility of the input substrate to anaerobic microorganisms. As a result of the pre-treatment is accelerating of the process of anaerobic fermentation and increasing of the yield of methane. Pre-treatment may be mechanical, biological, physical, chemical, or anyhow combined. This thesis is focused on the comparison of cumulative production of biogas produced by biomass coming from two different substrates (a mixture of poplar and willow leaves) originating from the area of Příbram and specific locations of Trhové Dušníky and Podlesí. These sites differ in the grade of contamination. Substrate from each site was modified in two ways, mechanically and also by thermal-pressure hydrolysis. To determine the cumulative production of biogas simple batch tests were applied. Measurements were carried out at regular intervals, initially from 2 to 3 times a day, then ones a day for 26 days. In the course of the experiment were analyzed for all substances, total dissolved solids content, organic content and the content of dissolved organic matter. In a course of the experiment, the total dissolved solids content, organic content ant the content of dissolved organic matter were analyzed for all substances. COD, ammonium-N content and pH were also determined. The largest cumulative biogas production was observed in mechanically treated sample from the site of Podlesí. The non-hydrolyzed substrate produced twice the amount of biogas than hydrolyzed substrate from the same locality. Mobilization of heavy metals is probably the cause of inhibition of anaerobic degradation of hydrolyzed substrate. The mobilization occurred after pre-treatment by thermal-pressure hydrolysis. Among the sites were also observed differences in the quantitative production of biogas. Both substrates from the site Trhové Dušníky showed lower biogas production. From the analysis of heavy metals and the results of the cumulative production of biogas is evident that the site Podlesí is more contaminated. But heavy metals, which are located in the biomass coming grom this area, have low mobility. Another cause of the inhibition process is likely to be higher hemicelluloses content in the substrate. Under the influence of the thermal - pressure hydrolysis hemicellulose cleaves the furan derivatives that have inhibitory effects. The ratio of polysaccharides (cellulose, hemicellulose and lignin), protein and lipid significantly affects the process of anaerobic digestion and efficiency of pretreatment. Thermal – pressure hydrolysis of biomass, containing large amount and high level of hazardous metals which have high mobility, negatively affects the anaerobnic digestion. Some methods of the preparation can increase the mobility of heavy metals and thus negatively affect the process. Knowledge of process and the consequences of pre-treatment is crucial in practice.