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One of the best ex situ conservation strategies for wild germplasm is in vitro conservation of genetic banks. The success of in vitro conservation relies heavily on the micropropagation or performance of the species of interest. In the context of global change, crop production challenges and climate change, we face a reality of intensified crop production strategies, including genetic engineering, which can negatively impact biodiversity conservation. However, the possible consequences of transgene presence for the in vitro performance of populations and its implications for biodiversity conservation are poorly documented. In this study we analyzed experimental evidence of the potential effects of transgene presence on the in vitro performance of Gossypium hirsutum L. populations, representing the Mexican genetic diversity of the species, and reflect on the implications of such presence for ex situ genetic conservation of the natural variation of the species. We followed an experimental in vitro performance approach, in which we included individuals from different wild cotton populations as well as individuals from domesticated populations, in order to differentiate the effects of domestication traits dragged into the wild germplasm pool via gene flow from the effects of transgene presence. We evaluated the in vitro performance of five traits related to plant establishment (N = 300): propagation rate, leaf production rate, height increase rate, microbial growth and root development. Then we conducted statistical tests (PERMANOVA, Wilcoxon post-hoc tests, and NMDS multivariate analyses) to evaluate the differences in the in vitro performance of the studied populations. Although direct causality of the transgenes to observed phenotypes requires strict control of genotypes, the overall results suggest detrimental consequences for the in vitro culture performance of wild cotton populations in the presence of transgenes. This provides experimental, statistically sound evidence to support the implementation of transgene screening of plants to reduce time and economic costs in in vitro establishment, thus contributing to the overarching goal of germplasm conservation for future adaptation.