Accessory, supernumerary, or-most simply-B chromosomes, are found in many eukaryotic karyotypes. These small chromosomes do not follow the usual pattern of segregation, but rather are transmitted in a higher than expected frequency. As increasingly being demonstrated by next-generation sequencing (NGS), their structure comprises fragments of standard (A) chromosomes, although in some plant species, their sequence also includes contributions from organellar genomes. Transcriptomic analyses of various animal and plant species have revealed that, contrary to what used to be the common belief, some of the B chromosome DNA is protein-encoding. This review summarizes the progress in understanding B chromosome biology enabled by the application of next-generation sequencing technology and state-of-the-art bioinformatics. In particular, a contrast is drawn between a direct sequencing approach and a strategy based on a comparative genomics as alternative routes that can be taken towards the identification of B chromosome sequences.