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Diego Godin and Co. appeared dumbfounded as the wrong anthem was played at the University of Phoenix Stadium in Glendale, Arizona.
The players stood silently throughout, while injured star Luis Suarez was spotted speaking to a team-mate on the sidelines.
Goalkeeper Fernando Muslera offered the most confusion, looking around awkwardly during the anthem.
That may have had an impact as Uruguay fell behind, with Alvaro Pereira heading in an own goal in the fourth minute.
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Uruguay will also face Venezuela and Jamaica in Group C.