Background: Transfer RNAs can participate in a variety of vital life activities. The gymnosperms with important ecological and economic values were the dominant and constructive species in the forest ecosystems in the North hemisphere. However, evolution and structural changes of chloroplast tRNA in gymnosperms remain largely unclear.Results: In this study, the nucleotide evolution, phylogenetic relationships and structural variations of 1,779 chloroplast tRNAs in gymnosperms were determined. The number and types of tRNA genes contained in the chloroplast genome of different gymnosperms were not much different, the average amount of tRNA contained was 33, and the occurrence frequency of various types of tRNA in gymnosperms was generally consistent. Nearly half of the anti-codons were absent. Molecular sequence variation analysis revealed the conservative secondary structure of tRNA. About a quarter of the tRNA genes were found to contain precoded 3’ CCA tails. A few tRNAs underwent novel structural changes that were closely related to their minimum free energy, these structural changes affected the stability of tRNA. Phylogenetic analysis showed that tRNAs have evolved from multiple common ancestors and tRNALeu was the most primitive type of tRNA. The transition rate of chloroplast tRNAs was higher than the transversion rate in gymnosperms. The tRNAs continuously have experienced more loss than duplication events during the evolutionary process. Conclusions: These findings provided novel insights into the molecular evolution and biological characteristics of the chloroplast tRNA in gymnosperms.