Verticillium wilt (VW) is a major constraint to cotton production in Australia and worldwide. The disease is caused by a soilborne fungus, Verticillium dahliae, a highly virulent pathogen on cotton. Commonly, V. dahliae is designated into two pathotypes: defoliating (D) and non-defoliating (ND), based on induced symptoms. In the previous two survey seasons between 2017 and 2019, stems with suspected VW were sampled for the confirmation of presence and distribution of D and ND pathotypes across New South Wales (NSW), Australia. A total of 151 and 84 VW-suspected stems sampled from the 2017/18 and 2018/19 seasons, respectively, were subjected to pathogen isolation. Of these, 94 and 57 stems were positive for V. dahliae; and 18 and 20 stems sampled respectively from the two seasons yielded the D pathotype isolates. Two stems from the 2017/18 season and one stem from 2018/19 season yielded both D and ND pathotype isolates. We also successfully demonstrated the co-infection of both pathotypes in pot trials, which was driven predominantly by either of the pathotypes, and appeared independent on vegetative growth, fecundity and spore germination traits. Our study is the first report of the natural co-occurrence of both D and ND pathotypes in same field-grown cotton plants in NSW, to which a challenge to the disease management will be discussed.