Would Trifles have the same impact if the characters were reversed--that is, the main characters were males?

The impact of the play depends on an understanding of the patriarchal culture that Mrs. Hale, Mrs. Peters, and Mrs. Wright functioned in, so a play in which a man murders his wife and his male neighbors withhold evidence would not have anywhere near the same impact as the play Trifles has. The fact that Mrs. Wright is a woman has everything to do with why she was treated the way she was and why she responded the way she did.

Mr. Wright, as the man of the house, made the decisions and had all the power in their relationship. Mrs. Wright was little more than a servant, trapped in their isolated home, without the ability to pursue her own interests. When she did attempt to express her wants by purchasing a canary, John Wright evidently broke the cage and killed the bird. This act of violence toward a defenseless creature suggests Mr. Wright was similarly abusive toward his wife. Mrs. Wright had no support system to help her escape from the trap she was in. If, for example, she had gone to Mr. Peters, the sheriff, and explained that she was afraid for her safety or her life because her husband had killed her bird, chances are she would have been ignored or even mocked. Mr. Peters and the attorney display condescension toward Mrs. Hale and Mrs. Peters, hinting at how any such complaint Mrs. Wright might have made about her husband would have been treated. 

Mrs. Wright's murder of her husband was an act of desperation that grew from the power disparity in her relationship with her husband and in her society. It is hard to imagine any comparable situation where the tables could be turned and a man would be so controlled by his wife. Although there certainly are controlling women, a husband in John Wright's time would still have enjoyed many options in his society simply by virtue of being a man--such as mobility and the support of the legal system and male-dominated social structure. Since the issues of abuse and power are central to the plot and conflict, and these are wrapped up in gender roles, the play could not have the same impact if the main characters were male rather than female.