River's quest for back-to-back titles will continue against Brazilian giant Cruzeiro in a two-legged round-of-16 clash starting in July.
After edging Argentine rival Boca in a dramatic final last season, River advanced to the knockout stage as runner-up to Internacional in Group A.
Meanwhile, two-time champion Cruzeiro – which last won the competition in 1997 having reached the final in 2009 – finished six points clear atop Group B.
Tuesday's (AEST) draw in Paraguay also has two other mouth-watering ties pitting Argentina against South American rival Brazil.
Six-time champion Boca will play Brazil's Paranaense – who won the Copa Sudamericana last year – after the two teams went head-to-head in Group G.
Boca finished two points clear of Paranaense, which won the first meeting 3-0 in Curitiba before the Argentine giants sealed top spot with a last-gasp 2-1 victory in Buenos Aires on matchday six.
The other Argentina-Brazil fixture will see Godoy Cruz take on Luiz Felipe Scolari's Palmeiras.
Copa Libertadores last-16 draw:
River Plate v Cruzeiro
Godoy Cruz v Palmeiras
Emelec v Flamengo
LDU Quito v Olimpia
Athletico Paranaense v Boca Juniors
Nacional v Internacional
Gremio v Libertad
San Lorenzo v Cerro Porteno