Introduction Cotton (genus Gossypium, Malvaceae) is a major natural fiber crop used by the textile industry and is an important oil crop, as well as a byproduct provider (seeds for cattle feed, short fibers to obtain cellulose for chemistry, etc.). Although the EU represents only 1.2% of the world's cotton production with 283,400 metric tons of raw cotton (Cotton Inc., 2017), the crop plays a determinant role in economic and social aspects of countries like Greece and Spain. The two countries account for 81.4% and 17.3% of the total European output, respectively (Eurostat, 2016). These regions provide cotton plants with high boll yields and high fiber quality due to the specialized, professional, and intensive farming practices being implemented. Nevertheless, there is a scarcity of research on European cotton, especially related to genetic diversity and molecular marker analysis. An enhanced understanding of both of these topics would be highly beneficial for developing new breeding strategies. The success of plant breeding programs mainly depends on the available genetic variability of crops. This variance depends on the genetic relationship and diversity between and within plant groups (Yu et al., 2012a; Abdellatif and Soliman, 2013). Thus, having a set of varieties that globally represent useful alleles is critical in any breeding program. It requires the management, conservation, and evaluation of valuable cotton materials while maintaining a continued supply of new germplasm. This plant breeding set could be made of new or old improved varieties with yield potential to our crop environment and other related wild materials or different species with desirable traits (Kaur et al., 2014). Nowadays, more than 95% of world cotton production comes from American cotton species Gossypium hirsutum L. and Gossypium barbadense L. (Upland and Pima cotton, respectively) (Abdurakhmonov et al., 2012). Upland varieties, which represent 90% of the total, are appreciated for their high yield and wide adaptability. Conversely, Pima varieties are cultivated for their fiber length and quality. Both New World species are allotetraploidic (AD) with 2n = 4x = 52 chromosomes (Fryxell et al., 1992) and can be cross-pollinated for commercial purposes in order to interchange traits via backcrossing or segregating