Lignin obtained in acidic conditions is a waste product in various technological processes like sulfite pulping, organosolv pulping, or bioethanol production. Knowing the structure of the lignin enables its use in high-value-added applications. In this paper, the lignin structure isolated from Pinus sylvestris L. and Populus deltoides × maximowiczii wood in acidic conditions was investigated. Two methods of lignin isolation (Klason method and a method using a sulfuric and phosphoric acid mixture) were compared. Additionally, lignin acetylation was performed. The lignin samples were analyzed using different in- strumental techniques, such as size exclusion chromatography (SEC), attenuated total reflection–Fourier transform infrared spectroscopy (ATR-FTIR), and scanning electron mi- croscopy (SEM). Based on the studies carried out, it was found out that the lignin isolated from pine and poplar wood in acidic conditions had a highly condensed structure. This was evidenced by the high-weight average molar mass of lignin (up to 118,700 g/mol) and the precipitates, aggregates, and agglomerates on its surface. Moreover, the characteristic signals of condensed lignin in ATR-FTIR analysis (band with wavenumber of 767 cm−1) and their decrease/disappearance (band that usually occurs with a wavenumber of about 814 cm−1) were observed. Lignin acetylation and analysis in the 0.5% LiCl/DMAc system have proven particularly effective in the case of the condensed poplar lignin. The beneficial effect of lignin acetylation was confirmed by SEM analysis. The high-molecular-weight condensed lignin, despite some of its problematic properties connected mainly with solu- bility, is a valuable substance that can be used for different applications (carbon fibers or as an additive for thermoplastic blends), which was confirmed by the studies in this paper and the findings of other scientists