The legal complaint was filed against Texas Tech University and professor Michael Dini by a student and the Liberty Legal Institute, a religious freedom group that calls Dini's policy "open religious bigotry."
"Students are being denied recommendations not because of their competence in understanding evolution, but solely because of their personal religious beliefs," said Kelly Shackelford, chief counsel for the institute.
The Department of Justice asked Texas Tech in Jan. 21 letter to respond to the allegations, university officials said.
Texas Tech spokeswoman Cindy Rugeley said that the university stands by Dini, and that his policies do not conflict with those of Texas Tech.
"A letter of recommendation is a personal matter between a professor and student and is not subject to the university control or regulation," Texas Tech Chancellor David Smith wrote in October in response to an earlier letter of complaint.
Dini, an associate professor who has been at Texas Tech for 10 years, said Wednesday he didn't know about a federal inquiry. He referred questions about his policy to a Web page that outlines it.
The Web page advises students seeking a recommendation to be prepared to answer the question: "How do you think the human species originated?"
"If you cannot truthfully and forthrightly affirm a scientific answer to this question, then you should not seek my recommendation for admittance to further education in the biomedical sciences," Dini writes.
The legal complaint began with Texas Tech student Micah Spradling, who withdrew from Dini's class and the university in the fall and enrolled at Lubbock Christian University after learning about Dini's policy.
Spradling, 22, wants to become a physician and said he needed a letter of recommendation from a biology professor but, as a creationist, he said he couldn't "sit there and truthfully say I believe in human evolution."
"It's a theory. You read about it in textbooks. I could explain the process, maybe how some people say it happens, but I could not have said ... I believe in it," Spradling said Wednesday. "I really don't see how believing in the evolution of humanity has anything to do with patient care or studying science."
Spradling re-enrolled at Texas Tech this semester, after obtaining a recommendation letter at the other school.
Dini writes that he has the policy because he doesn't believe anyone should practice in a biology-related field without accepting "the most important theory in biology."
"Good scientists would never throw out data that do not conform to their expectations or beliefs," he writes.
Dini also says he refuses to write letters of recommendation for students he doesn't know fairly well and those who haven't earned an "A" in one of his classes.
Department spokesman Jorge Martinez refused to not confirm or deny an investigation, citing department policy.