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The at-statement should work, e.g.:test.at<cv::Vec2f>[y][x][0] = 1.0 (or: test.at<cv::Vec2f>(y,x)[0] = 1.0).

Alternatively and much more convenient: instead of

cv::Mat test = cv::Mat::zeros(Img.rows, Img.cols, CV_32FC2);

use the templated Matrix-version, i.e.

cv::Mat2f test = cv::Mat2f::zeros(Img.rows, Img.cols);

then you don't need to use .at<> and the rest of your code should work as is.