The Marvel sequel, which broke a new US record for an April release last week, took $41.4m (£24.7m), adding to the previous week's haul of $96m (£58m).
Animated adventure Rio 2 was close behind, raking in $39m (£23.3m) on its debut, according to early estimates.
Low budget horror Oculus and football drama Draft Day were third and fourth.
Oculus took $12m (£7.1m) - more than double its $5m (£3m) budget - while Draft Day, starring Kevin Costner and directed by Ivan Reitman, took $9.8m (£5.8m) million.
Globally, The Winter Soldier also continued to fly, taking $60.6m (£36.2m) over the weekend.
The action movie has made nearly $477m (£285m) globally since its release internationally three weeks ago. That figure has already surpassed the $370m (£221m) total for 2011's Captain America: The First Avenger.
"The traditional rules just don't apply anymore. It's really about that Marvel brand,'' said Paul Dergarabedian, senior media analyst for box-office tracker Rentrak.
"Captain America can play in Peoria just as well as Hong Kong.''
Teen science fiction film, Divergent, took fifth position, adding $7.5 (£4.4m) in its fourth week to bring its cumulative total to $124.9m (£74.6m) million.
Lionsgate announced last week that the third instalment in the series will be split into two releases, one to open in March 2016 and one in March 2017. The second film will be released in 2015.
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