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Background: Metagenomics is the study of the genomic content of an environmental sample of microbes. Advances in the throughput and cost-efficiency of sequencing technology is fueling a rapid increase in the number and size of metagenomic datasets being generated. Bioinformatics is faced with the problem of how to handle and analyze these datasets in an efficient and useful way. One goal of these metagenomic studies is to get a basic understanding of the microbial world both surrounding us and within us. One major challenge is how to compare multiple datasets. Furthermore, there is a need for bioinformatics tools that can process many large datasets and are easy to use. Results: This paper describes two new and helpful techniques for comparing multiple metagenomic datasets. The first is a visualization technique for multiple datasets and the second is a new statistical method for highlighting the differences in a pairwise comparison. We have developed implementations of both methods that are suitable for very large datasets and provide these in Version 3 of our stand-alone metagenome analysis tool MEGAN. Conclusion: These new methods are suitable for the visual comparison of many large metagenomes and the statistical comparison of two metagenomes at a time. Nevertheless, more work needs to be done to support the comparative analysis of multiple metagenome datasets. Availability: Version 3 of MEGAN, which implements all ideaspresented in this paper, can be obtained from our website at:www-ab.informatik.uni-tuebingen.de/software/megan.