The aim of this study was to determine the traits that define the optical properties of the needles of four coniferous species: Picea abies, Picea omorika, Abies alba and Pinus sylvestris. The analysis included measurements of the needles for their morphological and anatomical aspects, reflectances at the upper and lower needle surfaces through their 280–880-nm spectra, and biochemical traits. The needles of these species differed significantly in the majority of morphological and anatomical traits, with the most pronounced differences seen for the thickness of the cuticle and epidermis, the needle width and thickness, the width of the central cylinder, and the position and density of the stomata. The reflectance spectra of the upper needle surface were very similar, while for the reflectance of the lower needle surface, P. omorika reflected light the most efficiently, followed by A. alba. The biochemical properties indicated significant differences in the amounts of UV-absorbing compounds, which were highest in P. sylvestris, and relatively low in A. alba and P. abies. The upper needle surface reflectance spectra were significantly affected by thickness of the cuticle, by pore width and by total mesophyll thickness, which explained 24%, 12% and 4% of the variability, respectively. The needle traits that explained the reflectance spectra variability of the lower needle surface were the hypoderm (28%), needle thickness (4%), density of stomata (28%), length of the outer pores (9%), and amount of UV-A-absorbing compounds (7%). Our data show that the needle reflectance spectra are primarily affected by the physical structure of the needles, and little by the needle biochemistry. This calls into question the methodologies for determination of the biochemical status of conifers based on their reflectance spectra.