Remote sensing (RS) is currently regarded as one of the standard tools used for mapping invasive and expansive plants for scientific purposes and it is increasingly widely used in nature conservation management. The applicability of RS methods is determined by its limitations and requirements. One of the most important limitations is the species percentage cover at which the classification result is correct and useful for nature conservation. The primary objective, carried out in 2017 in three areas of Poland, was to determine the minimum percentage cover from which it is possible to identify a target species by RS methods. A secondary objective of this research, related to the requirements of the method, was to optimize the set of training polygons for a target species in terms of the number of polygons and abundance percentage cover of the target species. Our method has to be easy to use, effective, and applicable, therefore the analysis was carried out using the basic set of rasters—the first 30 channels after the Minimum Noise Fraction (MNF) transformation (the mosaic of hyperspectral data from HySpex sensors with spectral range 0.4–2.5 µm) and commonly used Random Forest algorithm. The analysis used airborne hyperspectral data with a spatial resolution of 1 m to perform classification of one invasive and three expansive plants—two grasses and two large perennials. On-ground training and validation data sets were collected simultaneously with airborne data collection. When testing different classification scenarios, only the set of training polygons for a target species was changed. Classification results were evaluated based on three methods: accuracy measures (Kappa and F1), true-positive pixels in subclasses with different species cover and compatibility with field mapping. The classification results indicate that to classify the target plant species at the accepted level, the training dataset should contain polygons with a species cover ranging from 80–100%. Training performed only using polygons with a species characterized by a variable, but lower, cover (20–70%) and missing samples in the 80–100% range, led to a map which was not acceptable because of a high overestimation of target species. We achieved effective identification of species in areas where the species cover is above 50%, considering that ecosystems are heterogeneous. The results of these studies developed a methodology of field data acquisition and the necessity of synchronization in the acquisition of airborne data, and training and validation of on-ground sampling.