The rapid development of Next Generation Sequencing technologies leads to the accumulation of huge amounts of sequencing data. The scientific community faces an enormous challenge in how to deal with this explosion. Here we present a software tool, 'Gegenees', that uses a fragmented alignment approach to facilitate the comparative analysis of hundreds of microbial genomes. The genomes are fragmented and compared, all against all, by a multithreaded BLAST control engine. Ready-made alignments can be complemented with new genomes without recalculating the existing data points. Gegenees gives a phylogenomic overview of the genomes and the alignment can then be mined for genomic regions with conservation patterns matching a defined target group and absent from a background group. The genomic regions are given biomarker scores forming a uniqueness signature that can be viewed and explored, graphically and in tabular form. A primer/probe alignment tool is also included for specificity verification of currently used or new primers. We exemplify the use of Gegenees on the Bacillus cereus group, on Foot and Mouth Disease Viruses, and on strains from the 2011 Escherichia coli O104:H4 outbreak. Gegenees contributes towards an increased capacity of fast and efficient data mining as more and more genomes become sequenced.