Root architecture of prairie grasslands, which depends on plant phenology and edaphic conditions, strongly influences susceptibility to invasion by nonindigenous plant species. Field studies were conducted to compare in situ root growth patterns of warm-season (WS) and cool-season (CS) perennial grasses and musk thistle during a 2-yr period that included a drought in the second year. In 2 yr, CS grasses had the highest amount of roots (1,296 m roots m−2 [395 ft roots ft−2]) across shallow (0 to 28 cm [0 to 11 in.]), medium (28 to 56 cm), and deep (56 to 98 cm) depths with 65% occurring in the shallow depths. However, roots of WS grasses were always greater at deeper depths compared to roots of CS grasses. The amount of new roots in CS grasses was statistically different in 2011 (F2,43 = 33.3, P < 0.0001) at all depths for vegetative (April to May), inflorescence (June), and dormant (July to November) stages. In 2012, the amount of new roots in CS and WS grasses was statistically different (F2,60 = 81.7, P < 0.0001 and F2,37 = 8.0, P = 0.0013), respectively, for vegetative (April to May), inflorescence (May to June), and dormant (June to November) stages. For both years, the amount of new roots in the CS grasses showed an interaction between the three growth stages and three soil depths (F2,62 = 33.3, P < 0.0001 ; F4,60 = 18.6, P < 0.0001 ). From germination to senescence, the total amount of musk thistle roots was 298 m roots m−2, which was less than the CS (1,296 m roots m−2) and WS (655 m roots m−2) grasses. The largest proportion of new musk thistle roots (61%) (F2,42 = 40.4, P < 0.0001) occurred during the bolting stage (April to June) of the second year. These results show the difference in root distribution of two grass types and the niches that are created underground by extraneous conditions (e.g., drought) in WS grass stands that may contribute to the establishment of musk thistle, an invasive plant species in many North American regions.