Earlier, classical cytogenetics played a key role in taxonomic studies through identification of chromosome number and morphology. Similarly, the first identifications of polyploid species, and the analysis of relationships between different species from interspecific hybrids, were based on the observation of chromosome pairing during metaphase I of meiosis. Cytogenetics subsequently got a boost with the development of mapping and of next-generation sequencing technologies, enabling the development of modern molecular cytogenetics. In this chapter, we present the major impacts of molecular cytogenetics: shedding new light on genome organization and evolution as well as regulation of meiosis in the economically important genus Brassica and the tribe Brassicaceae. First, we present how comparative chromosome painting (CCP) using pools of Arabidopsis thaliana BAC clones is used to establish genome organization in diploid and polyploid species in conjunction with genotyping and sequencing data. This method complements phylogenetic analyses in establishment of the common ancestral genome and in the description of the three differentially fractionated Brassica ancestral subgenomes. Secondly, intergenomic relationships can be determined by BAC-fluorescent in situ hybridization (BAC-FISH) and genomic in situ hybridization (GISH); these techniques allow identification of the different genomes and chromosomes to quantify homologous and non-homologous pairing in haploids and hybrids, identifying structural rearrangements within allopolyploid species and between genomes in interspecific hybrids. Thirdly, meiosis and meiotic recombination in Brassica napus and its close relatives can be studied using antibodies developed against Arabidopsis proteins. From all these data, we show how molecular cytogenetics is essential for our understanding of genetics and genomics in the genus Brassica and how cytogenetics will undoubtedly play a significant role in the times to come.