Neck rot caused by the fungus Botrytis allii Munn. is one of the most devastating diseases of onions (Allium cepa L.), resulting in significant yield losses. This disease is latent in nature developing symptomless onion plants in the field with bulbs typically showing symptoms 1–2 months after harvest in the storage. Molecular studies were conducted to detect latent infections of Botrytis neck rot in the onion fields of Manitoba, Canada. Plant samples of onion cultivars ‘Redwing’ and ‘Pocono’ were collected every 10 days throughout the growing season, starting from planting until bulb harvesting during 2018, 2019, and 2020 from a research farm in Brandon, Manitoba, and plant samples of ‘Redwing’ were collected during 2019 and 2020 from a commercial vegetable farm in Portage La Prairie, Manitoba. The amplified DNA fragment of onion leaves and the neck region of collected samples were subjected to polymerase chain reaction using the Botrytis-specific primer pair BA2f/BA1r. Botrytis allii was detected on onion samples collected from the commercial farm as early as the end of June 2019 and 2020 when plants were at the 5–7 leaf stage. The majority of onion samples collected from the research farm also started testing positive for the pathogen from June (2019, 2020) and July (2018) onwards. This knowledge about the timing of infection in the field will be useful in helping farmers to develop and evaluate management strategies in the field, and also predict the storability and availability of quality bulbs for sale.