Plant roots grow into the soil for efficient acquisition of various nutrients, such as inorganic nitrogen, ammonium, and nitrate. A previous study has revealed the genetic diversity of foliar functions of Arabidopsis thaliana ecotypes in an environment containing ammonium; however, the function of roots remains unclear. This work focuses on the root system architecture (RSA) of Arabidopsis ecotypes to investigate the genetic factors regulating ammonium-dependent RSA changes. Arabidopsis ecotypes were grown on vertical agar medium containing ammonium as a major nitrogen source, and root growth and RSA were determined. Arabidopsis ecotypes showed differential sensitivity to ammonium. The shoot dry weight of some ecotypes decreased, whereas that of other ecotypes increased in ammonium medium. The RSA changes also varied among the different ecotypes in response to ammonium. The total root length, measured as the sum of primary root and lateral root length, of some ecotypes was reduced, whereas that of other ecotypes showed no significant difference in ammonium medium. Compared with lateral roots, the primary roots showed a sharp response to ammonium supply. Notably, the RSA showed a partial correlation with shoot dry weight in ammonium medium. Because Col-4 and Ler-0 showed opposite RSA responses to ammonium supply, these two ecotypes were selected for further genetic analysis. Quantitative trait locus (QTL) analysis of recombinant inbred lines of Col and Ler showed the involvement of several genetic factors in ammonium-dependent RSA changes. Moreover, QTL analysis revealed that the primary structure of nitrogen-related enzymes do not account for changes in RSA in response to ammonium supply.