The lengths of the actin (thin) filaments in sarcomeres directly influence the physiological properties of striated muscle. Although electron microscopy techniques provide the highest precision and accuracy for measuring thin filament lengths, significant obstacles limit their widespread use. Here, we describe distributed deconvolution, a fluorescence-based method that determines the location of specific thin filament components such as tropomodulin (Tmod) or probes such as phallacidin (a phalloidin derivative). Using Tmod and phallacidin fluorescence, we were able to determine the thin filament lengths of isolated chicken pectoralis major myofibrils with an accuracy and precision comparable to electron microscopy. Additionally, phallacidin fluorescence intensity at the Z line provided information about the width of Z lines. Furthermore, we detected significant variations in thin filaments lengths among individual myofibrils from chicken posterior latissimus dorsai and embryonic chick cardiac myocytes, suggesting that a ruler molecule (e.g., nebulin) does not strictly determine thin filament lengths in these muscles. This versatile method is applicable to myofibrils in living cells that exhibit significant variation in sarcomere lengths, and only requires a fluorescence microscope and a CCD camera.