Internal fruit rot of bell pepper is mainly caused by members of the Fusarium lactis species complex (FLASC), which contains multiple sequence types (STs). Less important causal agents are F. proliferatum and F. oxysporum. The relative pathogenicity of the FLASC STs and the other Fusarium species on a series of cultivars was tested via inoculation of pin-wounded fruit or via inoculation of the flowers, the natural infection pathway. Fusarium oxysporum produced the largest lesions on inoculated fruit; in general, yellow pepper cultivars were most susceptible. The presence of Fusarium inside fruit of flower-inoculated plants was evaluated after two weeks and at harvest. All Fusarium species were equally able to infect via flowers, but infection incidence was dependent on the cultivar. To determine the spore load of FLASC STs, F. oxysporum and F. proliferatum, we developed specific real-time PCR assays and applied them to samples taken with a Burkard volumetric spore sampler during the 2012 growing season. In general, spores were present in high numbers during spring and became less abundant later in the year. FLASC spores were the most numerous, explaining the higher incidence of this species in infected fruit. The real-time PCR detection technique was also used on surface samples. Horizontal surfaces usually contained large numbers of spores. Also, samples of organic residue that had dropped onto the floor were tested. In many cases, dried-up aborted fruits harboured very large numbers of FLASC spores, indicating that they might be an important source of inoculum.