Mixed cropping in short rotation coppice can be an alternative to monocultures. To design optimized mixtures, field trials are needed. Poplar, as an economically important and fast-growing species, and black locust, as a nitrogen-fixing species, are promising candidates for such studies. RNA sequencing (RNA-seq) was used to monitor effects of mixed and pure cultivations on the gene expression of poplar along with growth measurements during 2017 and 2018. Both biomass production and leaf transcriptomes revealed a strong competition pressure of black locust and the abiotic environment on poplar trees. Gene expression differed between the two study sites and pure and mixed stands. Shading effects from black locust caused the downregulation of photosynthesis and upregulation of shade avoidance genes in mixed stands in 2017. As a result of higher light availability after cutting black locust, plant organ development genes were upregulated in mixed stands in 2018. Drought conditions during the summer of 2018 and competition for water between the two species caused the upregulation of drought stress response genes in mixed stands and at the unfavorable growing site. Further investigations are required to discover the mechanisms of interspecific competition and to develop stand designs, which could increase the success and productivity of mixed plantations.