More than 500 people were believed to be on board both trains.
Esther Padilla, spokesperson for the government of Castilla-La Mancha region, told TVE that one of the trains - managed by the Iryo company - was carrying 146 passengers plus one baby, while the Renfe-managed train had 397 passengers on board.
A former government minister was also among those reported missing.
The Spanish Confederation of Business Organizations (CEOE) posted on X that, Fátima Báñez - who heads the organization's foundation - was on board one of the trains involved in the crash.
Local leader Emiliano García-Page, president of Castilla-La Mancha, said "at least 30" people are now confirmed to have died.
Speaking separately to Spanish media, he warned the death toll could increase further.
Eighty people are being treated for injuries, according to authorities, with 50 of the casualties said to be in a serious condition.
Spanish Prime Minister Pedro Sánchez said he was "shocked by the serious train accident in Toledo".
"I remain in constant contact with the Minister of Transport, and with the president of Castilla-La Mancha," Sánchez said on X, adding that emergency services were working on the ground "to assist those affected and ensure their safety, in coordination with all the involved administrations".
"My affection, solidarity, and support to all the victims and their families."
Sánchez and other Spanish politicians have cancelled all public engagements on Wednesday, including campaigning for next month's parliamentary elections.
Spain's royal family posted on X that it was "shocked and concerned" by the accident and expressed condolences for the relatives of the deceased and those affected by the crash.
A spokesperson for the EU said the bloc was ready to help Spain if needed.
A huge emergency response is under way with fire crews and helicopters at the scene.
Spanish news agency have reported that hospitals in the area have suspended planned surgeries as they deal with an influx of patients from the crash.
A blood transfusion centre in Madrid has also appealed for "urgent" donations. In a post on X, Centro de Transfusión said it made the request "in the face of a possible sudden increase in care needs".
It reported that facilities in Toledo, Ciudad Real and Alcázar de San Juan have cancelled procedures as casualties from the crash begin to arrive.
This is the second high-speed rail tragedy in Spain this year.
In January a high-speed train derailed in southern Spain, its rear carriages going into the path of a state-run Renfe train, claiming the lives of 46 people.