It had been proposed that long-spored isolates of Verticillium from crucifers were amphihaploid interspecific hybrids between V dahliae and V albo-atrum. The original aims of this thesis were to construct artificial hybrids between these two species and analyse the hybrids through molecular, genetic and pathological means. It was shown that limited recombination is possible between nit mutants of each species. Through pathogenicity testing on oilseed rape it was shown that long-spored amphihaploid isolates could be differentiated from haploid V dahliae isolates. However, during this work it was found that of the non-V dahliae-like supposed 'parent' was unlike any isolate of V albo-atrum found to this day, and this species was unlikely to be involved in the hybridisation events that produced amphihaploid isolates of Verticillium. The projects main aims were revised to further characterise the long-spored isolates molecularly and through pathogenicity testing so their 'parental' origins were better understood. Molecular analysis of the rRNA gene V-region and ITS showed that a disease outbreak in cauliflowers in Belgium was caused by isolates molecularly similar to those from oilseed rape in Europe, and that isolates from Lucerne in Iran were V albo-atrum. This thesis reports the first direct evidence that long-spored amphihaploid isolates of Verticillium from crucifers are interspecific hybrids through amplification of the 5S rRNA IGR and the β-tubulin gene. Sequence analysis of these along with the mitochondrial cytochrome B gene proved that V albo-atrum was not a 'parent' of the amphihaploid isolates, but the 5S rRNA IGR supports the further division of Verticillium amphihaploids AFLP group B, to form a third group y, represented by isolates from horseradish in Illinois, USA. Although the 'unknown parent' involved in the hybridisation events has yet to be found, through the results from this thesis it would now be possible to identify it molecularly.