The hosts took the lead in the 25th minute through former Chelsea forward Alvaro Morata, who finished off a quick counter-attack.
Portugal equalised late on in the 82nd minute. Joao Cancelo set up substitute Ricardo Horta, who scored his first international goal, eight years after winning his only other cap.
Fernando Santos' side, who won the first edition of the competition in 2019, host Switzerland next. He does, however, face numerous selection dilemmas, with many players failing to impress against their great Iberian rivals.
One of those players was Manchester United's Bruno Fernandes, who continued his poor run of form with an ineffective display against Spain. The Portugal playmaker scored just ten times in 46 appearances for the Red Devils across the campaign
Despite scoring twice in his nation's crucial World Cup play-off victory over North Macedonia in March, the 27-year-old was hooked after 81 minutes. Supporters took to Twitter to slate Fernandes for his performance, with some even suggesting he shouldn't be going to Qatar later this year:
Spain's experienced side fail to see off Portugal
La Roja's six-game winning run came to an end as they were forced to settle for a point in their opening game of the 2022 Nations League.
Luis Enrique decided to go with an experienced team to face their neighbours, with the likes of Sergio Busquets, Jordi Alba and Cesar Azpilicueta all starting. Despite having 61% possession and attempting over 200 more passes than their opponents, the hosts failed to see the game out.
Spain have failed to win a World Cup knockout game since winning the tournament in 2010. Enrique is determined to put that dismal record to rest in the Middle East later this year, with many players treating the winter tournament as their last opportunity to win football's biggest prize.
Portugal also named an experienced lineup, with Joao Moutinho and 39-year-old centre-half Pepe starting. Meanwhile, Cristiano Ronaldo came on just after the hour mark, although the five-time Ballon d'Or winner failed to get on the scoresheet.