The National Redemption Front (NRF) is an alliance of Darfur rebels and political parties that reject the peace deal struck in May. It was formed in the Eritrean capital, Asmara, last week and attacked Hamrat al-Shaikh, 200km from Khartoum, on Monday.
Lam Akol, the Sudanese foreign minister, said: "If they form a movement in Asmara and come and fight against Sudan and we have asked Asmara to mediate in problems in the east, then that does not augur well for peace."
Eritrean-Sudanese relations had improved in recent months, and Asmara sent an ambassador to Khartoum last month. Asmara is mediating in talks intended to end a simmering 10-year-old conflict in eastern Sudan.
Most of the opposition groups have either signed agreements with Khartoum or are in peace negotiations.
But Eritrea's hosting of the new rebel alliance has raised a question over its ability to mediate neutrally, Akol said.
"This is why we are seeking clarification so we can get an answer to that question - we told them we need an immediate answer," he said.
The Eritrean embassy in Khartoum declined to comment.
Monday's attack in North Kurdufan, which neighbours Darfur, prompted a hasty response from Sudan's armed forces, which sent bombers to repulse the offensive.
The NRF said a two-year-old humanitarian ceasefire was dead, the first time a rebel group has openly denounced the truce, although it had in any case been largely ignored by all parties.
Majzoub al-Khalifa, the Sudanese presidential adviser, also accused Chad of supporting the NRF, in comments carried in state-owned press.
Chad has played host to many of the rebel commanders involved in Monday's attack. Sudan has also been home to Chadian insurgents bent on overthrowing the president, Idriss Deby.