## 1 Introduction  
Cotton (_Gossypium hirsutum_ L) is among the most important sources of natural plant fiber and seed oil [1, 2]. Over the last five decades, the global cotton industry has seen dramatic changes, with production nearly quadrupling from 6.6 million tons in 1950/51 to a record 26.3 million tons in 2004/05. The average growth rate in global production has been approximately 2.5% per year. However, yields leveled off during the 1990s due to disease, pesticide resistance, and economic disruption. Yields began to rise again in the late 1990s as seed varieties improved and living modified (LM) cotton was introduced [3]. LM cotton is one of the major LM crops, along with soybean, maize, and canola. Cotton production is vulnerable to biotic stresses such as insect attacks and abiotic factors such as temperature, drought, and salinity [4, 5, 6, 7]. LM crops are widely used in agriculture to improve crop quality by introducing new traits, including insect resistance, herbicide tolerance, higher yields, and resistance to abiotic and biotic stresses [8, 9, 10]. LM cotton with certain traits such as insect resistance and herbicide tolerance was also developed. Herbicide-resistant LM cotton employs _epsps, bar, pat, dmo,_ and _aad-12_ genes to withstand herbicides that would otherwise destroy the crop along with the targeted weeds [11, 12, 13, 14, 15]. The use of insect-resistant LM cotton introduces _Bacillus Huntingiensis (Bt)_ toxins, such as crystal toxin (Cry) and vegetative insecticidal protein (Vip), which can reduce costs and environmental impact by discontinuing or decreasing the use of chemical insecticides [16, 17, 18, 19]. In 2018, LM cotton plantations covered 24.9 million hectares (75.7%) of the 32.9 million hectares of global cotton cultivation, with LM cotton possessing traits for insect resistance, herbicide resistance, or both, accounting for 72.9%, 3%, and 24% of the global cotton cultivation, respectively [20].  
Although the cultivation of LM crops is not permitted in South Korea, a total of 32 LM cotton events, including single genes and stacked traits, have been approved for food, feed, and processing (FFP). A total of 100,000 tons of LM cotton was imported in 2008, and since then, the rate of import has steadily increased every year [20]. However, there is growing concern about the unintentional release of LM cotton into the natural ecosystem in South Korea. As a result, the Ministry of Environment (MOE) and National Institute of Ecology (NIE) has been conducting an environmental monitoring and post-management initiative for living modified organisms (LMOs) since 2009. The MOE detected unintentionally released LMO plants in certain areas of the country from 2009 to 2013, and PCR analysis was performed on suspicious samples using the 35S promoter and _nos_ terminator-specific primer pairs to determine the presence of transgenic events.  
The aim of this study was to investigate unintentionally released LM cotton in natural environments in South Korea, classifying their collection sites and characterizing their transgenic traits using advanced analysis techniques. The study's findings support the need for an LMO management system to reduce the risks posed by released LMOs and efficiently preserve the natural ecosystem in South Korea.