Due to the rapid development of China’s economy, the demand for wood is steadily increasing. Eucalyptus species have been introduced in large quantities because of their fast growth, strong adaptability, and wide utility. To understand the phenological changes in introduced Eucalyptus in its new range, we carried out a field investigation to examine leaf functional and chemical defense traits of three introduced species (E. saligna, E. grandis and E. robusta) over latitudinal and altitudinal gradients in southern China. We sampled multiple stands of each species, and measured the leaf physical characteristics (e.g., leaf width, leaf thickness, and specific leaf area [SLA]), leaf nitrogen (N) and phosphorus (P) content, and phenolic compounds. We found that many functional traits (e.g., leaf size and thickness) decreased at lower latitudes, especially in E. grandis, possibly to reduce heat and water loss under higher temperatures. In E. grandis, we found that leaf P was lower at higher latitudes and altitude, and phenolics increased with elevation, while in E. robusta, both leaf N and P decreased with altitude. These findings suggested that both species were more conservative in resource allocation, with E. grandis possessing enhanced chemical defenses in response to the conditions experienced at higher elevations. In addition, we found the tree populations at the northern range limit of E. robusta had lower SLA, suggesting a more conservative growth strategy, In contrast, small populations in the northern part of the ranges of E. grandis had higher SLA, indicating range expansion at the edge of the species’ geographic distribution. Overall, it is particularly important to consider intraspecific trait differences across wide geographic areas when studying the spread of invasive species in the new range.