Due to their relatedness to Arabidopsis thaliana (Arabidopsis), the cultivated Brassica species represent the first group of crops with which to evaluate comparative genomics approaches to understanding biological processes and manipulating traits. We have constructed a high-quality binary BAC library (JBo) from genomic DNA of Brassica oleracea var. alboglabra, in order to underpin such investigations. Using the Arabidopsis genome sequence and clones from the JBo library, we have analysed aspects of gene conservation and microsynteny between a 222 kb region of the genome of Arabidopsis and homoeologous segments of the genome of B. oleracea. All 19 predicted genes tested were found to hybridize to clones in the JBo library, indicating a high level of gene conservation. Further analyses and physical mapping with the BAC clones identified allowed us to construct clone contig maps and analyse in detail the gene content and organization in the set of paralogous segments identified in the genome of B. oleracea. Extensive divergence of gene content was observed, both between the B. oleracea paralogous segments and between them and their homoeologous segment within the genome of Arabidopsis. However, the genes present show highly conserved collinearity with their orthologues in the genome of Arabidopsis. We have identified one example of a Brassica gene in a non-collinear position and one rearrangement. Some of the genes not present in the discernible homoeologous regions appear to be located elsewhere in the B. oleracea genome. The implications of our findings for comparative map-based cloning of genes from crop species are discussed.