Cotton GHB614 × LLCotton25 was produced by conventional crossing. The EFSA GMO Panel previously assessed the two single cotton events GHB614 and LLCotton25 and did not identify safety concerns. Integrity of the inserts was retained in the two-event stack cotton. No differences requiring further food and feed safety assessment were identified in the compositional analysis of cotton GHB614 × LLCotton25 except for a higher level of gossypol. The EFSA GMO Panel further assessed this difference and considers that it is of no safety relevance for animals and humans. Expression analysis and safety assessment of the newly expressed proteins identified no concerns regarding their potential toxicity and allergenicity. No indications of safety issues regarding the overall allergenicity of cotton GHB614 × LLCotton25 were identified. There are no indications of an increased likelihood of establishment and spread of feral cotton plants. Considering the scope of the application, potential interactions of cotton GHB614 × LLCotton25 with the biotic and abiotic environment were not considered a relevant issue. Environmental risks associated with an unlikely but theoretically possible horizontal gene transfer from cotton GHB614 × LLCotton25 to bacteria have not been identified. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of cotton GHB614 × LLCotton25.