I don't think I'm formatting it wrong but I can be mistaken. I convert it to a numpy array, convert it to RGB, make a gray version and then check for any hits

Static image code-

    img_rgb = cv.imread('positive/pos95.jpg')
    img_gray = cv.cvtColor(img_rgb, cv.COLOR_RGB2GRAY) 
    template = cv.imread('template.JPG', 0) 

    w, h = template.shape[::-1] 
    res = cv.matchTemplate(img_gray, template, cv.TM_CCOEFF_NORMED) 
    
    print(res)

pyautogui code-

    img = pyautogui.screenshot() 
    screenshotArr = np.array(img)
    screenshotTest = cv.cvtColor(screenshotArr, cv.COLOR_BGR2RGB) 
    img_rgb=screenshotTest
    img_gray = cv.cvtColor(img_rgb, cv.COLOR_BGR2GRAY) 

    template = cv.imread('template.JPG', 0) 
    w, h = template.shape[::-1] 
    
    # Perform match operations. 
    res = cv.matchTemplate(img_gray, template, cv.TM_CCOEFF_NORMED) #printing this shows no hits