Nitrogen (N) is the primary macronutrient required for plant growth and survival; consequently, massive amount of N fertilizers are used to enhance crop yield. One of the ways of reducing the N input is by breeding for N-efficient crops. In maize, genotypes with well distributed root system have high potential to adapt to soils with limited nutrient availability. The aim of this study was to use association analysis to identify polymorphisms within root genes controlling root development in maize seedlings grown under contrasting N levels. For this purpose, association study (AS) panel consisting of 74 diverse set of inbred maize lines were screened for root and shoot traits under two contrasting nitrogen levels (low and high nitrogen) in two independent experimental conditions. Allele re-sequencing of RTCL, RTH3, RUM1 and RUL1 genes related to root development was carried in the AS panel using cycle sequencing procedure. Association analysis was carried between individual polymorphisms and root traits while controlling for spurious associations due to population structure and kinship relations. Linkage disequilibrium decayed rapidly at all the candidate gene loci studied. Based on the SNP’s identified, lines within the AS panel were grouped into several haplotypes. Associations analysis revealed several polymorphisms within root genes putatively associated with the variability in root and shoot development under contrasting N levels. Thus considerable allele polymorphisms present within RTCL, RTH3, RUM1 and RUL1 genes could be utilized to improve root architecture in maize. Polymorphisms within root genes plausibly associated with root traits could be used to develop functional markers that allow identifying maize lines with contrasting expression of NUE for rapid screening of the trait in early growth stages.