Nitrogen is one of the limiting nutrients for plant growth. Biological fixation in microorganisms transforms atmospheric nitrogen into forms which are accessible to plants. Nitrogen-fixing bacteria or diazotrophs are generally free living but are able to form a symbiotic association with plants. The most abundant diazotrophs are known as rhizobia, mainly constituted of Alphaproteobacteria. Frankia species are the most recently discovered nitrogen-fixing bacteria which form a symbiotic association with plants of 8 different genera, compared to rhizobia which are able to form a symbiotic association only with legumes. Comparing symbiotic-related genes which are needed for the initiation and maintenance of symbiosis and nitrogen-fixation, relationships have been detected which point to horizontal gene transfer and the ability to acquire the aforementioned functional features. Analyzing the codon usage in diazotrophs of (non)symbiotic associations with plants, I researched the functional characteristics of genes unique to nitrogen-fixing organisms as well as differences among them. According to differences in codon usage I found genes which are related to symbiotic nitrogen-fixation and postulated the existence of horizontal gene transfer of these genes among different bacterial species. I classified the inspected organism into groups, based on the functional profiles created, which bear similarities with their lifestyle and/or phylogeny based on 16S rRNA.