Centre forward Eder, who scored the winning goal in the Euro 2016 final against France, was among 10 players from that squad omitted from Santos's list.
Other unlucky players included midfielders Andre Gomes and Renato Sanches, forward Nani and defender Eliseu.
Cristiano Ronaldo, Portugal's all-time leading scorer and most-capped player, headed the list for the European champions, who face Spain, Morocco and Iran in a tricky first round group.
Other stalwarts included goalkeeper Rui Patricio, central defenders Pepe (35) and Bruno Alves (36), midfielders William Carvalho and Joao Moutinho and flamboyant forward Ricardo Quaresma.
"It was extremely difficult to choose the 23. As a professional, I have to make decisions and I know that I'm not going to please everyone," Santos told a news conference.
"But, on the personal side, it hurts me to leave out players who took part in the European championship, and others who took part in the qualifiers and helped the team get this far.
"It was, of course, difficult to leave out Eder, Nani and others who wrote the most brilliant page in the history of Portuguese football. But I have to pick the ones who best complete the jigsaw."
Russian-based midfielder Manuel Fernandes, who is 32 and has won 12 caps, was one of those to be included alongside Napoli left back Mario Rui.
"I can't say whether this squad is stronger than the one which played at the Euros," Santos said. "But I have absolute confidence in this squad just as I did in the other one."