Conyza spp. are broadleaf weeds that occur in many crops but are also common in non-crop systems such as roadsides and railways. Conyza have selected for glyphosate resistance along railway tracks in southern Spain due to the misuse of this herbicide and the high seed dispersal rate of these species. Twenty-three samples of the genus Conyza (11 Conyza canadensis and 12 Conyza bonariensis) were collected from the margins of railways in different routes of the Andalusia railway network running adjacent to nearby crop fields. The glyphosate resistance level of Conyza populations was evaluated through GR₅₀ (herbicide rate causing 50% growth reduction) and resistance factor (RF) values in every population collected. The highest GR₅₀ were 1851.2 g a.e. ha^-1 (RF = 52.53) in C. canadensis (Malaga–Cordoba route) and 1972.4 g a.e. ha^-1 (RF = 35.20) in C. bonariensis (Seville–Cordoba route), and the lowest were 46.9 g a.e. ha^-1 (RF = 1.33) in C. canadensis (Seville–Cordoba route) and 23.2 g a.e. ha^-1 (RF = 0.41) in C. bonariensis (Seville–Cordoba route). The results showed that, among all the C. canadensis populations collected, 18.2% were glyphosate-resistant (RF > 10), 45.5% showed a tendency to develop resistance (RF = 2.5–5), and 36.4% were susceptible (RF < 2.5). Of the 25% of C. bonariensis populations that had resistance to glyphosate, 16.7% had moderate resistance (RF = 5–10) and 58.3% were susceptible. This study found that there are already glyphosate-resistant Conyza spp. along the railway network in southern Spain. This could lead to possible seed exchange between the railway and adjacent places. Therefore, it is vital to consider the railway network when planning control measures against resistance.